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WO2019035972A1 - Immunisations séquentielles avec des particules de type virus env du vih-1 - Google Patents

Immunisations séquentielles avec des particules de type virus env du vih-1 Download PDF

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Publication number
WO2019035972A1
WO2019035972A1 PCT/US2018/000247 US2018000247W WO2019035972A1 WO 2019035972 A1 WO2019035972 A1 WO 2019035972A1 US 2018000247 W US2018000247 W US 2018000247W WO 2019035972 A1 WO2019035972 A1 WO 2019035972A1
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hiv
env
virus
subtype
vlp
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Baozhong Wang
Teena MOHAN
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Georgia State University Research Foundation Inc
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Georgia State University Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • HIV-1 human immunodeficiency virus type-1
  • HIV vaccine would have a massive influence in curtailing new infections, but a potentially licensable vaccine candidate remains elusive.
  • some of the key milestones have been achieved in the development of an HIV-1 vaccine, including the RV144 trial which showed an unprecedented 31.2% reduction in HIV incidence, the prospects for a highly effective and affordable HIV-1 vaccine seem remote.
  • the HVTN 100 trial has been carried out to evaluate the adapted versions of the RV144 trial designed specifically for the population of South Africa. If several key immune response targets are met, it will set the stage for a far larger Phase III efficacy trial (HVTN 702) with the potential to lead to licensure.
  • HIV-1 evolves rapidly within the host, resulting in the accumulation of diverse HIV- 1 quasi-species.
  • the envelope gene encodes a 160-kDa glycoprotein designated HIV-1 envelope (Env) that hides conserved CD4 and co-receptor binding sites with an evolving shield of glycans, variable immunodominant loops, and conformational masking.
  • HIV-1 Env embedded in the viral membrane, is the only target of neutralizing antibodies, thus presenting a moving target to the host immune system. What is needed are new vaccines or immunization strategies that can more effectively adapt to the rapid mutation and evolution of HIV-1.
  • methods of generating broadly neutralizing antibodies against a virus and/or method of immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from a virus or viruses for which protection is sought over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round.
  • the virus is selected from the group of viruses consisting of Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Zika virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St.
  • viruses consisting of Herpes Simplex virus- 1,
  • VLP virus like particle
  • the antigen of the VLP is selected from the HIV antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat), regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif), for example, an HIV envelope (env) VLP such as a chimeric trimeric HIV env VLP.
  • each chimeric trimeric HIV-1 envelope VLP from first to last VLP is HIV-1 subtype C, HIV-1 subtype B, HIV-1 subtype D, HIV-i subtype A, and HIV-1 subtype E.
  • administration occurs one month after the previous administration.
  • viral administration strategies comprising the sequential administration of one or more viral antigens to a subject over two or more
  • each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round.
  • kits immunizing a subject against a virus comprising two or more clade variants each of one or more viral antigens.
  • Figures 1A, IB, 1C, ID, IE, IF, 1G, 1H, II, 1J and IK show the chimeric HIV Env gene construct and characterization of VLPs.
  • (la) Schematic representation of HIV- 1 Env gene. The signal peptide-encoding sequences of HIV Env genes from subtypes A, B, C, D, and E were replaced by the honeybee melittin signal peptide sequence using overlapping PCR, to increase Env glycoprotein expression in SF9 insect cells. Conformation-stabilized trimeric Env proteins were made by the addition to C-terminus of a trimeric form of a leucine zipper sequence,
  • GCN4pii to stabilize Env trimers. HIV Env gene containing melittin and GCN4pii were cloned into the pFastBac-1 transfer vector;
  • VLPs were crosslinked with BS3 at various concentration to confirm the oligomeric state of the HIV Envs as described in the materials and methods, (lg) Total Env content in VLPs. A quantitative ELISA was done to determine the Env glycoprotein content in VLPs, using recombinant HIV- 1 S 162 Env as a calibration standard. Unrelated (influenza) VLPs were used as negative control group. Results showed the data of three independent experiments and represented as mean ⁇ SD; (lh) PGT145 monoclonal antibody binding assay. The assay was performed as mentioned in material and methods. Unrelated (influenza) VLPs were used as negative control group.
  • Results showed the data of three independent experiments and represented as mean ⁇ SD; (li) HIV-1 Env VLPs binding to glycan-dependent PGT126 and PGT128. Unrelated antibody (anti-histidine) and Gag VLP were used as negative control groups in the assay. Results showed the data of three independent experiments and represented as mean ⁇ SD; (lj) TEM pictures of prepared VLPs; and (lk) Zeta potential of the representative VLPs.
  • Figure 2 demonstrates the results of cell-based ELISA to determine the endpoint titers against respective HIV Env (autologous) antigens as described in materials and methods.
  • the IgG endpoint titers were measured against HIV-1 Env of subtype A, subtype B, subtype C, subtype D, or subtype E expressed on HE 293T cells.
  • Cells infected with unrelated plasmid and pre-immune sera have been used as negative control group.
  • Rabbits were immunized with PBS, Gag VLPs, single (subtype B) Env VLPs, a mixture of various Env VLPs, and sequential immunization of HIV Env VLPs through i.m. route of vaccination.
  • Figure 3a and 3b show Con-S Env-specific antibody titers and IgGASCs.
  • the figure represents antigen-specific (3a) serum IgG endpoint titers; and (3b) IgGASCs, at the 3rd week of last vaccination using the recombinant Con-S Env protein as coating antigen.
  • Rabbits were immunized with PBS, Gag VLPs, single (subtype B) Env VLPs, a mixture of various Env VLPs, and sequential immunization of HIV Env VLPs through i.m. route of vaccination. The highest dilution which gave an OD450 two-fold higher than that of the naive group without dilution was designated as the antibody endpoint titer.
  • Figures 4A, 4B and 4C show antigen-specific serum IgG subtype levels.
  • Figure represents Env-specific IgG subtype endpoint titers in serum samples (4a) IgG2a; (4b) IgG2b; and (4c) IgGl subtypes.
  • Rabbits were immunized with PBS, Gag VLPs, single (HIV subtype B) Env VLPs, mixture of various Env VLPs, and sequential immunization of HIV Env VLPs through i.m. and in. routes of vaccination. Animals were primed at week 0; and further repetitive and sequential doses were given at every 4 th week.
  • Antibody avidity assays were conducted with immune sera collected at the 3rd week of last vaccination from the animals immunized with (5a) Gag VLPs; (5b) single (subtype B) Env VLPs; (5c) a mixture of various HIV Env VLPs; and (5d) sequential immunization of HIV Env VLPs.
  • the avidity index values were determined by measuring the resistance of antibody-envelope glycoprotein complexes in the ELISA by treatment with 1.5 M NaSCN.
  • Figure 6 shows sera neutralization assay.
  • Heat map of ID50 values obtained with the sera tested individually against a panel of 32 pseudoviruses from tier 1, 2, and 3 of various HIV subtypes.
  • the figure shows the ID50 values in the animal groups vaccinated with PBS, Gag VLPs, single (subtype B) Env VLPs, a mixture of various VLPs, and sequential immunization of Env VLPs. Results were compared with the ID50 values of the sera collected before vaccination (pre-immune sera), Gag only (non-Env) VLP, and unrelated (influenza H7N9) pseudovirus. The reciprocal of sera dilution necessary to achieve 50% neutralization was reported as the ID50 value.
  • Figures 7A, 7B, 7C, 7D, 7E, and 7F show ADCC assay results. ADCC was assessed using a standard 51Cr release assay. Figure showed ADCC in serum samples collected from immunized animals with (7a) single (HIV subtype B) Env VLPs; (7b) mixture of various VLPs; (7c) sequential i.m. immunization; (7d) sequential i.n. immunization; while (7e) and (7f) showed ADCC in nasal and vaginal wash samples, respectively, of animals immunized using sequential i.n. immunization approach.
  • FIG. 8A, -8B, 8C, and 8D show serum and mucosal antibody levels. Rabbits were immunized repetitively with PBS, Gag (non-Env) VLPs, single Env (clade B) VLPs, or sequentially with different HIV-1 Env VLPs. Sera and mucosal washes were collected at one- week pre- (preimmune samples) and 3-week after the last vaccination.
  • Serum (IgG, IgGl, IgG2a, and IgG2b) and mucosal IgA (nasal and vaginal) levels were determined against Con-S Env (consensus) and various Env (autologous) antigens using indirect ELISA and cell-based ELISA, respectively.
  • the HIV Con-S Env protein (200ng/well) and the transfected HEK293T cells (5 xlO 4 cells/well) were used as coating antigens.
  • HEK293T cells were transfected with various HIV-1 Env clones and negative control plasmids as mentioned in the methods.
  • the transfected cells were seeded at a density of 5xl0 4 cells/well and later fixed by 80% acetone prior to sample inoculation. After washing and blocking, serially diluted sera/mucosal washes were added, and the color was developed.
  • the figure represents antigen-specific endpoint titers of (8 A) serum IgG; (8B) nasal IgA; (8C) vaginal IgA; and OD 450 values at 1 :100 fixed dilution of (8D) serum IgG subtype. The highest dilution which gave an OD 45 o 2-fold higher than that of the naive group without dilution was designated as the antibody endpoint titer. Results show the data of three independent experiments and results were expressed as the mean ⁇ SD.
  • FIGS 9A, 9B, 9C, and 9D show antibody avidity indices and neutralization titers towards different pseudoviruses.
  • Antibody avidity and neutralization assays were conducted with immune sera and mucosal lavages collected from the ammals immunized with various vaccine formulations.
  • the avidity indices were determined by measuring the strength of antibody-envelope glycoprotein complexes in ELISA by treatment with 1.5M NaSCN.
  • the avidity indices were calculated from the ratio of the absorbance value obtained with 1.5M NaSCN treatment to that observed with PBS treatment multiplied by 100.
  • the figures 9A, 9B, and 9C represent the antibody avidities of the animals immunized sequentially with various HIV Env VLPs towards various pseudoviruses (tier 1, 2, and 3) in the (9 A) sera; (9B) nasal wash; and (9C) vaginal wash samples. Results showed the data of three independent experiments and results were expressed as the mean ⁇ SD.
  • the figure 9D shows the heat map of ID 50 obtained with the nasal wash tested against a panel of 32 pseudoviruses using TZM-bl neutralization assay in the animal groups vaccinated with various vaccine formulations. Results were compared with the ID 5 o of the samples collected before vaccination (preimmune nasal wash) and unrelated (influenza H7N9) control pseudovirus.
  • FIGS 10A and 10B show CD4 and CD8 T cell proliferation.
  • PBMCs were collected from the individual animals of each vaccination group after the last immunization and stimulated in vitro with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin
  • the figure shows the FACS analysis of (10A) CD3 + CD4 + and (10B) CD3 + CD8 + double positive T cell populations in various groups i.e. Gag VLPs, single Env VLPs, and sequential immunization group. The results were shown as one of the representative experiment.
  • FIG. 11 A, 1 IB, 11C, 1 ID, 1 IE, and 1 IF show Thl/Th2 cytokine secretions and evaluation of cytokine-secreting cells.
  • the levels of Thl/Th2 cytokines such as (11 A) IFN- ⁇ ; (1 IB) IL-2; (11C) TNF-a; and (1 ID) IL-6, were estimated from the cultured PBMCs' supernatant using quantitative ELISA.
  • PBMCs were in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone.
  • FIG. 1 IE shows IFN-y-producing cell number in the in vitro stimulated PBMCs' culture.
  • the ELISPOT assay was used for quantitating IFN- ⁇ - producing cell spots as described in the methods.
  • Figure 1 IF represents the results of antigen- specific IFN- ⁇ producing CD4 and CD8 T cells, quantified by an ICS assay as mentioned in the methods.
  • CD4/CD8 T cells were gated with IFN- ⁇ expression at specific antigen concentration for measuring IFN- ⁇ producing CD4 or CD8 T cell populations. Results were expressed as the mean ⁇ SD of each group of animals from three independent experiments.
  • Figures 12A and 12B show flow cytometric detection of CD4 and CD8 T cell degranulation.
  • the upper half of the figure (12 A) represents CD4 + CD107a + T cell population in the group of animals immumzed with Gag VLPs, single Env LPs, or sequentially immunized group of animals.
  • the lower half of the figure (12B) shows CD8 + CD107a + T cell subset in the group of animals immunized with various vaccine formulations.
  • Freshly isolated PBMCs from each group after the last vaccination were cultured and in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone and later stained with appropriate fluorescent antibodies as described in the methods.
  • the results were expressed as a percentage of CD4 + /CD8 + CD107a + cells and the results represented one of the
  • FIG. 13 A, 13B, and 13C show cytokine polyfunctionality and CD8 TCR avidity.
  • the figure shows the cytokine polyfunctionality or cytokines' response profile of (13 A) CD4 and (13B) CD8 T cells from each vaccination group.
  • CD4 or CD8 T cell populations were gated and single, double or triple cytokine expressing cell percentage enumerated. The percentage cytokine expression of each animal was averaged within the group and is expressed as a pie-chart of relative proportions of the total cytokine expressing cell population.
  • the figure (13C) demonstrates the CD8 TCR avidities or the IFN- ⁇ secretions by CD8 T cells in various vaccine formulations using ICS protocol as described in the methods.
  • CD8 T cells were gated with IFN- ⁇ expression and was measured as percent (%) maximum response at different antigen concentrations. All the results were expressed as the mean ⁇ SD from three independent experiments.
  • Ranges can be expressed herein as from “about” one particular value, and/or to
  • bnAbs broadly neutralizing antibodies
  • bnAbs broadly neutralizing antibodies
  • These bnAbs are formed through successive cycles of antibody mutation, selection and virus escape in some of HIV- 1 infected individuals. This process usually takes at least a couple of years, too long to offer any natural resistance to infection.
  • New technologies have contributed to the identification of many bnAbs capable of potently inhibiting multiple HIV-1 isolates from different clades. Because endogenous neutralizing antibodies are so slow to arise and do not always have a sufficient repertoire of recognized epitopes to keep pace with viral mutations, these antibodies do not help infected individuals to control the virus.
  • bnAbs can provide protection when they are in the host immune system prior to an exposure, such as being induced by a vaccination approach.
  • many attempts to generate bnAbs have uniformly failed.
  • the methods disclosed herein succeed where others have failed. Accordingly, in one aspect, disclosed herein are methods of generating broadly neutralizing antibodies against a virus. It is understood and herein contemplated that the disclosed methods of generating broadly neutralizing antibodies against a virus can be used to immunize a subject against infection with a virus. Thus, in one aspect, also disclosed herein are methods of immunizing a subject against a viral infection.
  • immunizing a subject against a virus comprising sequentially administering to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more viral antigens from one or more viruses against which neutralizing antibodies or immunological protection are sought.
  • the route of administration of the VLP can affect the immune response generated. Routes such as intramuscular, intraperitoneal, intravenous, and subcutaneous, can have greater effect on systemic immunity, whereas mucosal routes of administration such as intranasal, oral, rectal, or vaginal can be used to elicit an more mucosal immune response.
  • methods of generating mucosal antibodies against a virus comprising sequentially administering to a subject one or more viral antigens from a virus for which protection is sought over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding administration round, and wherein each
  • administration occurs intranasally, orally, vaginally, and/or rectally.
  • Antigen means any native or foreign substance that is capable of eliciting an immune response.
  • the one or more antigen will elicit an antibody, plasma cell, plasmablast, or B-cell response (including, but not limited to memory B cells and activated B cells).
  • antigens can include but are not limited to peptides, polypeptides, and/or proteins from a virus (i.e., viral antigens).
  • Viral antigens can include any peptide, polypeptide, or protein from a virus and can be part of a live virus, a live attenuated virus, an inactivated or killed virus, a virus-like particle (VLP).
  • VLP virus-like particle
  • the antigen can be an antigen from a virus selected from the group consisting of Herpes Simplex virus- 1 , Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Zika virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St.
  • a virus
  • the antigen can comprise HIV antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat), regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif).
  • group specific antigen Gag
  • Env Envelope glycoprotein
  • Polymerase Polymerase
  • Tat transactivator
  • Rev virion proteins
  • viral protein R Vpr
  • viral protein U Vpu
  • viral infectivity factor Vif
  • methods of generating broadly neutralizing antibodies against a virus and or immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from a virus against which immunological protection or broadly neutralizing antibodies is sought; wherein the virus is HIV (for example HIV-1).
  • kits for generating broadly neutralizing antibodies against a virus and/or immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from a virus against which immunological protection or broadly neutralizing antibodies is sought; wherein the virus is HIV (for example HIV-1); and wherein the antigen of the VLP is selected from the HIV (including HIV-1) antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat),regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif).
  • HIV including HIV-1 antigens consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, Negative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat),regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral
  • the disclosed antigens can be presented as part of a composition, such as a vaccine
  • the term "vaccine” refers to an agent, including but not limited to a peptide or modified peptide, a protein or modified protein, a live virus, a live attenuated virus, an inactivated or killed virus, a virus-like particle (VLP), or any combination thereof, that is used to stimulate the immune system of an animal or human in order to provide protection against e.g., an infectious agent.
  • the vaccine can comprise an HIV-1 env viruslike particle.
  • Vaccines frequently act by stimulating the production of an antibody, an antibody- like molecule, or a cellular immune response in the subject(s) that receive such treatments.
  • vaccines can be administered in a pharmaceutically acceptable carrier either prophylactically or therapeutically.
  • the VLPs can assume a more "native like” conformation of a viral antigen (such as, for example, HIV-1 env which natively forms a trimeric form).
  • a viral antigen such as, for example, HIV-1 env which natively forms a trimeric form
  • the conformation of the antigen is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or combination thereof such as a dimer of dimers, a trimer of dimers, a dimer of trimers or other combination
  • the VLP can assume said conformation including homologous or heterologous permutations.
  • the VLP's can be constructed comprising trimeric HIV-env.
  • said trimeric HIV-env can be single (i.e, homologous) or chimeric (either heterologous including monomers in the timer form different clades or strains or monomers that comprise components from two or more clades, variants, or strains) constructs of HIV env.
  • a trimeric form of a leucine zipper sequence such as, for example GCN4pii
  • the leucine zipper sequence can be operationally linked to C-termini of the HIV antigen (for example, a trimeric Env).
  • sequential immunizations with several Env variants can shape the B-cell maturation and germinal centers (GCs) towards bnAb responses.
  • the sequential immunizations can occur, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more times.
  • methods of generating broadly neutralizing antibodies against a virus and/or immunizing a subject against a viral infection comprising sequentially administering to a subject one or more viral antigens from one or more viruses against which immunological protection or broadly neutralizing antibodies is sought sequentially over two or more administration rounds (for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more rounds.
  • each successive round can comprise one or more HIV antigens to a different HIV clade variant (for example, HIV subtype A (such as, for example, 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5), HIV subtype B (such as, for example, SF162, 6535.3, WIT0416.33, RHPA4259.7, CAAN5342.A2, and/or TRJ04551.58), HIV subtype C (such as, for example, ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12), HIV subtype D (
  • HIV subtype A such as, for example, 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5
  • HIV subtype B such as,
  • each successive round can comprise one or more HIV antigens to a different HIV clade variant, wherein the HIV clade variants comprise 92UG037.8 (HIV subtype A), SF162 (HIV subtype B), ZM53M.PB12 (HIV subtype C), 92UG021.16 (HIV subtype D), 93TH976.17 (HIV subtype E).
  • one or more subsequent rounds can provide to the subject a clade variant of the one or more antigens that is the same as at least one preceding administration round.
  • the antigen from the subsequent administration round that is of the same clade variant than one or more preceding administration rounds can be the same or a different antigen than previously administered.
  • the order of the immunization can be based on any rationale deemed appropriate by the administering physician.
  • the order of clades administered can be A,R,C,D,E; C,B,D,A,E; B,A,C,D,E; C,A,B,D,E ; A,C,B,D,E; B,C,A,D,E;
  • the order of clades for each round can be HIV subtype C, HIV subtype B, HIV subtype D, HIV subtype A, and then HIV subtype E.
  • the immunization order can further comprise HIV subtype F and HIV subtype G. It is understood and herein contemplated that the order for one antigen clade variants that is administered concurrently with a second antigen does not have to be the same order.
  • the VLP vaccine being administered can comprise strain variants from multiple different strains of the clade subtype.
  • contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade A strains including, but not limited to 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5; where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade B is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade B strains including, but not limited to SF162, 6535.3,
  • contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade C strains including, but not limited to ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12; where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade D is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu
  • a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade F is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade F strains including, but not limited to 93BR019.4, 93BR019.10, 93BR020.17, and/or 93BR029.2; and/or where a VLP comprising a Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from HIV Clade G is administered, contemplated herein is the administration of the Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif antigen from one or more Clade G strains including, but not limited to 93BR019.4, 93BR019.10, 93BR020.1
  • VLPs and methods of generating broadly neutralizing antibodies methods of generating mucosal antibodies, and/or methods of immunizing a subject against a virus wherein the VLPs and/or VLPs being administered comprises strain variants of each viral antigen for one or more successive subtype immunizations.
  • Chimeric antigens are genes or proteins comprising regions, domains, and/or features from two or more different strain or subtype (i.e., clade) variants of the same genes or protein or a domains of different genes or proteins of the same or different virus, strain, or subtype.
  • strain or subtype i.e., clade
  • antibodies, methods of generating mucosal antibodies, and/or methods of immunizing a subject against a virus can be chimeric antigens. It is understood and herein contemplated that the disclosed chimeric antigens can comprise any combination of strains and/or subtypes of gpl20, gp41 or the variable or constant domains of gpl20 of any of the viral strains or subtypes (i.e., clades) disclosed herein.
  • the chimeric antigen can be a chimeric Env comprising at least one variable domain from a first HIV strain variant (such as, for example, HIV subtype A (such as, for example, 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5), HIV subtype B (such as, for example, SF162, 6535.3, WIT0416.33, RHPA4259.7, CAAN5342.A2, and/or TRJ04551.58), HIV subtype C (such as, for example, ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12), HIV subtype D (such as, for example, 92UG021.16, 92UG013.70, 93ZR001.3, 92UG021.9, 92UG024.2, and/or 92UG021.16),
  • the chimeric Env can comprise at least one variable domain from a first strain variant and a constant domain from a second strain variant wherein the strain variants for the constant and variable domains are from different subtypes.
  • a VLP comprises a chimeric antigen in a dimeric, trimeric, tetrameric, pentameric, hexameric, or heptameric form
  • one or more of the monomeric components of the multimeric chimeric antigen can be derived from a different virus, strain, or subtype.
  • the chimeric Env can be a trimeric Enc wherein one, two, or three of the monomer Envs forming the trimer comprises a gpl20 variable domain that is from a different strain of HIV, but the same subtype relative to the variable domains of the other Envs in the trimer, but the gpl20 constant domains are from the same strain of HIV.
  • the chimeric Env can be a trimeric Enc wherein one, two, or three of the monomer Envs forming the trimer comprises a gpl20 variable domain that is from a different strain and subtype of HIV relative to the variable domains of the other Envs in the trimer, but the gpl20 constant domains are from the same strain of HIV.
  • the HIV Env gene, gpl60 protein product is proteolytically cleaved in the endoplasmic reticulum into two subunits, gpl20 and gp41.
  • a multimeric chimeric antigen can comprise at least one, two, three, four, five, six, or seven gpl20 subunits from Envs of different strains of the same or different subtypes relative to the other gpl20 Envs in the multimer while the gp41 is from Envs of the same strain.
  • the chimeric Env can be a trimeric Env comprising at least one gpl20 from HIV a first HIV subtype (such as, for example ZM53M.PB12 of subtype C) and at least one gpl20 from a second HIV subtype (such as, for example SF162 or subtype B) and potentially at least one gpl20 from a third HIV subtype (such as, for example, 92UG021.16 of subtype D); wherein the gp41 subunits of each monomer are from the same HIV strain (such as, for example, ZM53M.PB12).
  • one or more of the gpl20 Envs can be from different strains of the same subtype.
  • the chimeric Env can be a trimeric Env comprising at least one gpl20 from HIV a first HIV strain of an HIV subtype (such as, for example, subtype C) and at least one gpl20 from a second HIV strain of the same subtype (such as, for example, ZM197M.PB7 of subtype C) and potentially at least one gpl20 from a third HIV strain of the same subtype (such as, for example, DU172.17 of subtype C); wherein the gp41 subunits of each monomer are from the same HIV strain (such as, for example, ZM53M.PB12).
  • a first HIV strain of an HIV subtype such as, for example, subtype C
  • a second HIV strain of the same subtype such as, for example, ZM197M.PB7 of subtype C
  • a third HIV strain of the same subtype such as, for example, DU172.17 of subtype C
  • the gp41 subunits of each monomer are from the same
  • successive rounds of administration can be accomplished at any rate sufficient to allow promote affinity maturation of B cells and antibodies and ultimately provide broadly neutralizing antibodies.
  • each antigen administration i.e., each successive round
  • each antigen administration occurs about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 weeks, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the preceding round .
  • the period between immunization rounds can be the same or different between rounds.
  • the time period between successive rounds can increase or decrease.
  • the clade variants of the immunizing antigen (such as, for example, an HIV env VLP) can be administered every 4 weeks.
  • the disclosed immunization methods can be used prophylactically and
  • the antigen such as, for example, an HIV env VLP
  • administration of the antigen can occur prior to exposure to the virus against which protection is sought.
  • the immunization methods disclosed herein are being used in a therapeutic manner, the first VLP is administered after exposure to the virus against which protection is sought.
  • the generation of broadly neutralizing antibodies against a viral antigen can be an active immunization of the subject in which protection against the virus is sought with the broadly neutralizing antibodies developing in the subject.
  • the immunization of a subject with broadly neutralizing antibodies developed outside of the subject is immunized (i.e., a passive immunization).
  • the methods of immunization can include generating broadly neutralizing antibodies against an antigen in a first subject such as a human, non-human primate, rodent (such as, for example, a mouse, rat, guinea pig, rabbit, hamster, or gerbil), canine, bovine, feline, or equine; obtaining antibodies from the first subject; and administering the broadly neutralizing antibodies from the first subject to a second subject desiring/needing protection against the virus form which the antigen is derived.
  • the first subject can be from the same or different species than the second subject.
  • the first subject can be a genetically modified animal that produces human or humanized antibodies.
  • VLPs virus-like particles
  • the VLPs disclosed herein are unique and novel.
  • VLPs comprising an HIV (including HIV-1) antigens selected from the consisting of the group specific antigen (Gag), Envelope (Env) glycoprotein, egative regulatory factor (Nef), Polymerase (Pol), transactivator (Tat),regulator of expression of virion proteins (Rev), viral protein R (Vpr), viral protein U (Vpu), and viral infectivity factor (Vif).
  • HIV specific antigen can be any Env, Gag, Nef, Pol, Tat, Rev, Vpr, Vpu, and/or Vif derived from any of the HIV clades or strains disclosed herein, including, but not limited to HIV subtype A (such as, for example, 92UG031.4, RW92009.14, 92UG031.7, 92UG037.8, RW92009.17, and/or RW92020.5), HIV subtype B (such as, for example, SF162, 6535.3, WIT0416.33, RHPA4259.7, CAAN5342.A2, and/or TRJ04551.58), HIV subtype C (such as, for example, ZM53M.PB12, ZM197M.PB7, DU172.17, ZM135M.PL10A, ZM249M.PL1, and/or DU156.12), HIV subtype D (such as, for example, 92UG021.16, 92UG01
  • the VLPs can assume a more "native like" conformation of a viral antigen (such as, for example, HIV-1 env which natively forms a trimeric form).
  • a viral antigen such as, for example, HIV-1 env which natively forms a trimeric form
  • the conformation of the antigen is a dimer, trimer, tetramer, pentamer, hexamer, heptamer, or combination thereof such as a dimer of dimers, a trimer of dimers, a dimer of trimers or other combination
  • the VLP can assume said
  • the VLP can further comprise a dimeric, trimeric, tetrameric, pentameric, hexameric, or heptameric form of a leucine zipper sequence such as, for example GCN4pii, to the C-termini of an HIV protein (for example, Env) cytoplasmic sequences to produce conformation-stabilized multimeric (such as, for example trimeric Env) proteins.
  • a leucine zipper sequence such as, for example GCN4pii
  • an HIV protein for example, Env
  • cytoplasmic sequences to produce conformation-stabilized multimeric (such as, for example trimeric Env) proteins.
  • VLP constructs comprising HIV Env gene and GCN4pii, wherein the GCN4pii is functionally linked to the C-termini of Env.
  • the VLP can also be further modified to increase expression in a particular cell-type such as, for example, removing the native signal peptide encoding sequences of the antigen and replacing said sequence with signal peptide sequence that will be more readily expressed in the infected cell (for example, honeybee melittin signal peptide for expression in SF9 cells).
  • a particular cell-type such as, for example, removing the native signal peptide encoding sequences of the antigen and replacing said sequence with signal peptide sequence that will be more readily expressed in the infected cell (for example, honeybee melittin signal peptide for expression in SF9 cells).
  • VLP constructs comprising the signal peptide sequence of honeybee melittin, the HIV env gene, and GCN4pii, , wherein the GCN4pii is functionally linked to the C-termini of Env, for example, as depicted in Figure 1 A.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • parenterally e.g., intravenously
  • intramuscular injection by intraperitoneal injection
  • transdermally extracorporeally, topically or the like
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector.
  • compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, .D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al, Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • stealth and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research; 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation.
  • receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • compositions including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • compositions may also include one or more active ingredients such as antimicrobial agents, antiindElarnmatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable..
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryi amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389.
  • a typical daily dosage of the antibody used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • viral immunization strategies for generating broadly neutralizing antibodies and/or developing protective immunity against a virus in a subject comprising the sequential administration of one or more viral antigens to a subject over two or more administration rounds; wherein each successive administration round provides to the subject a different clade variant of the one or more antigens than any preceding
  • kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
  • the kits can include any reagent or combination of reagent discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
  • the kits could include one or more antigens and the clade variants for each antigen.
  • kits for generating broadly neutralizing antibodies against HIV, mucosal antibodies against HIV, and/or immunizing a subject against HIV comprising VLPs of two or more clade variants each of one or more HIV antigens (for example, HIV-1 Env VLP for HIV-1 subtype C, HIV-1 subtype B, HIV-1 subtype D, HIV-1 subtype A, HIV-1 subtype E, HIV-1 Env VLP for HIV subtype F, and/or HIV-1 Env VLP for HIV subtype G) and/or chimeric HIV antigens.
  • the kit can further comprise instructions to administer clade variants of the one or more viral antigens sequentially.
  • the kit can comprise HIV-1 Env VLP for HIV-1 subtype A, HIV-1 subtype B, HIV-1 subtype C, HIV-1 subtype D, HIV-1 subtype E, and instructions to administer the VLP to a subject in the order of subtype C, subtype B, subtype D, subtype A, and subtype E.
  • Example 1 Sequential immunizations with a panel of HIV-1 Env viruslike particles coach immune system to make broadly neutralizing antibodies a) Results:
  • HIV-1 Env incorporated VLPs showed high levels of Env content and retained their physical and functional properties:
  • Modified Env gene constructs were generated by replacing the original signal peptide encoding sequences with the honeybee melittin sequence and adding a trimeric form of leucine zipper sequence, GCN4pii to the C-termini of Env cytoplasmic sequences to increase Env glycoprotein production and to express conformation-stabilized trimeric Env proteins, respectively (Fig. la).
  • GCN4pii trimeric form of leucine zipper sequence
  • GCN4pii to the C-termini of Env cytoplasmic sequences to increase Env glycoprotein production and to express conformation-stabilized trimeric Env proteins, respectively
  • SF9 Spodoptera frugiperda
  • Insect cells have been used since the early years of HIV-1 vaccines and because these cells carry out many post-translational modifications including high- mannose type N- and O-linked glycosylations, resulting in glycoproteins with similar antigenicity and functionality as of mammalian system.
  • Cell-based ELISA results demonstrated that Env glycoproteins were expressed at the surfaces of recombinant baculovirus (rBV)-infected SF9 insect cells, as indicated by the enhanced OD450 value when compared to the control cells infected with unrelated (influenza) rBVs (Fig. lb).
  • VLPs with their existing physical and functional properties.
  • the sequential-immunization group we observed that subtype-specific IgG endpoint titers were at its maximum.
  • the sequential immunization group developed IgG with endpoint titers an order of magnitude greater (p ⁇ 0.001) than the mixture of various VLPs and other control groups.
  • single Env VLPs group demonstrated significantly (p ⁇ 0.01) higher sera reactivity towards subtype B Env antigen.
  • the mixture of various VLPs group showed moderate levels of antibody levels towards each of the HIV-1 Env antigens than other negative control groups.
  • Thl and Th2 type of immunity respectively:
  • An antibody response can result in changes in the distribution of IgG subclasses, and depend on the nature of the antigen, frequency and duration of the antigenic stimulation, and routes of immunization.
  • single Env VLPs or physical mixture of various VLPs dramatically increased antigen-specific IgG subclasses when compared to other control groups.
  • Rabbits vaccinated with single Env VLPs or mixture of various VLPs showed much higher IgG2a (Fig. 4a) and IgG2b (Fig. 4b) levels when compared to IgGl responses, indicating that these groups induce Thl -biased antibody responses.
  • Serum antibodies from the sequentially immunized group showed increased avidity indices towards Al, A3, A5, A6, Bl, B4, CI, C4, Dl, and D3 HIV-1 pseudostrains (Fig. 5d). These results demonstrate that sequential- immunizations induced antibody responses with higher avidity indices to most of the
  • pseudotyped virions tested including some of the tier 3 pseudostrains and might also indicate that sequential exposure of different Env sharing conserved epitopes to the immune system is important in triggering bnAb responses. (7) Sequential i.m. immunizations with various HTV-1 Env
  • VLPs enhanced bnAb responses.
  • the heat map shows the 50% inhibitory dilution (ID50), the reciprocal of sera dilution necessary to achieve 50% neutralization, from the various vaccine groups.
  • ID50 50% inhibitory dilution
  • Sera from the Gag VLP-immunized group did not show detecta- ble ID50 ( ⁇ 10) against any of the pseudoviruses (data not shown).
  • ID50 of 150-200 especially against Bl, B4, and B6.
  • ID50 in the mixture of various VLPs immunization group, we observed ID50 in a range of 200 ⁇ 00 against Bl, B3, B5, C5, C6, and D2
  • pseudoviruses which represents a 22% (7 out of 32 pseudoviruses) neutralization potency.
  • Low levels of ID50 ( ⁇ 20) against subtype E and F pseudoviruses were found while no detectable ID50 was observed against subtype G pseudoviruses.
  • results showed that immune sera of the sequential immunization group neutralized -70% of the pseudoviruses (23 out of 32 pseudoviruses).
  • the sequentially immunized group has shown greater ID50 values against some of the tier 3 HIV pseudostrains also such as A5, A6, and B6. Animals in this group showed ID50 in the range of 100-150 against subtype F and G pseudoviruses also, even though the vaccine formulation did not contain sub- type F and G HIV-1 Env antigens.
  • the negative control groups including pre-immune sera, non-Env (Gag) VLPs, and unrelated (influenza H7N9) pseudovirus showed no background neutralization.
  • ADCC antibody dependent cellular cytotoxicity
  • nnAbs non-neutralizing antibodies
  • serum and mucosal samples were analyzed for their ADCC activity using 51Cr release assay.
  • the mixture of various VLPs group (Fig. 7b) showed serum antibody responses with significantly (p ⁇ 0.05) higher ADCC than single Env VLP (Fig. 7a) or other control groups while the sequential i.m. immunized animals showed very low serum ADCC activity (Fig. 7c). Animals immunized sequentially through i.n.
  • HIV-1 Despite many efforts since HIV-1 first identified in 1983, and even after encouraging results from the RV144 trial, HIV-1 continues to infect almost one million individuals each year, a potentially licensable vaccine candidate remains a decade away.
  • Major challenges for an HIV preventing vaccine that can elicit protective bnAb responses are the genetic diversity, mutability of HIV-1 target epitopes and structural properties of a viral Env that hides conserved CD4 and co-receptor binding sites by modulating signature glycan motifs. These challenges were overcome by the design of novel Env immunogens that resemble the natural viral Env spikes and can trigger the selection and expansion of germline precursor and intermediate memory B- cells to recapitulate B-cell ontogenies associated with the generation of a bnAb response.
  • Equally important for vaccine development is the identification of innovative vaccination strategies that can mimic the natural process of infection to drive somatic hypermutation and B- cell maturation against heterologous primary virus envelopes.
  • Env immunogens intended to induce bnAbs can mimic the native structure as closely as possible. Thus, it is fair to display Env in situ, such as VLPs, in the optimal approach to induce bnAb responses.
  • a vaccination regimen recapitulate the dynamic process of antigenic changes of an HIV infection to induce bnAbs is needed.
  • bnAb responses to HIV Env-enriched VLPs were evaluated in rabbits when immunized through a sequential immunization pattern. Sequential i.m.
  • Env VLPs were used from various HIV-1 subclades, using this diversity to generate bnAb responses both by presenting new epitopes as escape variants and by fostering the response against more conserved epitopes.
  • the findings indicate that sequential administrations of several Env VLPs can stimulate a stronger bnAb response than repetitive deliveries of a cocktail of these VLPs or single Env VLPs.
  • a properly designed sequential vaccination scheme with different variants of Envs offers hope to manipulate antibody development which can more efficiently produce bnAbs.
  • Antibody avidity has been used as a measure of functional maturation of the humoral immune response and represents the combined binding affinities of a variety of antibodies and their multivalent antigen. Class switching, affinity maturation, and somatic hypermutation that occur during B-cell maturation generate high-affinity antibodies of different subclasses. Simultaneously, as most antigens have a diversity of antigenic determinants per protein molecule, an increased avidity can be a consequence of progressive appearance and accumulation of classes of antibodies, each specific for a distinctly different antigenic determinant.
  • HIV-1 Env VLPs administered through sequential i.n. immunization enhanced antigen-specific cellular immunity.
  • sequential i.n. immunized rabbits showed sustainable and significant increases in lymphocytes proliferation and CD8 + T-cell cytotoxicity.
  • some high levels of IFN- ⁇ , IL-2, and IL-6 in the culture supernatant showed a Thl type of immune response.
  • Preliminary evidence and different non-human primate studies indicate that a vigorous T-cell response, including IFN- ⁇ production, is an immune correlation of protection from HIV-1/SIV infection. The sequential i.n.
  • immunization can involve activating macrophages and lymphocytes, for the enhancement of antigen-specific T-cell functions. Collaboratively, sequential i.n. immunization preferentially augmented Thl -cell response resulting in high levels of IFN- ⁇ and IL-2. Another possible explanation is that mucosally administered Env VLPs can be presented to T-cells and stimulate the transcription of cytokine genes in activated T-cells or the enhancing cytokine levels can be promoted by local innate inflammatory and systemic adaptive immune responses.
  • T-cells can exist that can interact with IgG2a positive B-cells, perhaps by recognizing B-cell surface IgG2a. Alternatively, T-cells can induce greater Ig heavy chain gene switching in virgin B-cell clones responding to antigen exposure. Thus, additional activation signals given by T-cells at the time of B-cell triggering may promote the process of gene switching in such a way as to make it more likely that distal constant region genes, such as IgG2a, are expressed.
  • SF9 insect cells ATCC, Manassas, VA, USA
  • 293 T-cells ATCC, Manassas, VA, USA
  • TZM-bl cells HIV-1 Env clones of various strains, soluble human CD4, HIV-1 SF162 gpl20, Gag recombinant protein pr55, HIV Con-S Env peptide pool, HIV Con-S Env protein, goat anti-HlV-1 Env polyclonal antibody, monoclonal antibodies e.g. VRCOl, PGT126, PGT145, F425 B4al, and bl2 were acquired from the NIH AIDS Research and Reference Reagent Program.
  • rBVs of HIV-1 Env (subtype A-E) proteins were generated. Five different rBVs using Env clones were made from each of the HIV-1 subclades for sequential immunizations: 92UG037.8 (subtype A), SF162 (subtype B), ZM53M.PB12 (subtype C), 92UG021.16 (subtype D), and 93TH976.17 (subtype E).
  • the rBVs expressing HIV-1 Env glycoproteins from different subtypes or Gag protein were generated by using the Bac-to-Bac insect cell protein expression system (Life Technologies, Carlsbad, CA, USA).
  • HIV-1 Env (Env/Gag) VLPs were produced by co-infection of SF9 cells with rBVs expressing trimeric Env and Gag protein at the optimum MOI of 4: 1. Gag VLPs produced by infection of SF9 cells with rBVs expressing Gag protein alone were used as a control. At 60 h post-infection, VLPs were concentrated from the cell culture supernatant by porous fiber filtration using AKTA Flux (GE Healthcare, Uppsala, Sweden) and purified using sucrose density gradient centrifugation.
  • AKTA Flux GE Healthcare, Uppsala, Sweden
  • Env glycoproteins were infected with rBVs expressing Env at the MOI of 4 PFU/cell. After fixing the cells, Env surface expression was determined by ELISA using polyclonal goat anti-HIV-1 Env followed by horseradish peroxidase (HRP)-conjugated antibodies. The OD 450 nm was read with an ELISA reader (BioTek, Winooski, VT, USA), which was proportional to the surface expression of Env glycoproteins. Furthermore, the glycoprotein-CD4 binding capability was measured to examine whether the Env glycoprotein expressed on the cell surface were folded correctly.
  • HRP horseradish peroxidase
  • SF9 cells were infected with rBVs expressing Env at optimum MOIs and later incubated with soluble human CD4 (5 ⁇ ).
  • the amount of bound CD4 was analyzed by FACS with FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using FITC labeled goat anti-human CD4 antibody.
  • the VLP protein concentration was determined by Micro BCA protein assay and contamination of endotoxins in VLPs were excluded by Limulus amebocyte lysate (LAL) assay (Thermo Fisher Scientific, Waltham, MA, USA).
  • Gag and Env glycoprotein profiles in VLPs were analyzed by SDS-PAGE followed by Western blot using anti-Gag and anti-HIV-1 Env polyclonal antibodies, respectively.
  • a quantitative sandwich ELISA was also done to determine total Env glycoprotein contents in VLPs, using recombinant Con-S Env gp 120 as a calibration standard.
  • the morphology, size distribution, and zeta potential of prepared VLPs were determined by TEM (Zeiss, Oberkochen, Germany) and zetasizer (Malvern, Massachusetts, MA, USA).
  • Sequential immunization regimen containing a panel of VLPs of various Envs as mentioned below (groups 5 and 6) were compared with repetitive homologous-immunizations of individual (subtype B) Env VLPs (group 3) and a mixture of various VLPs of various Envs (group 4).
  • PBS group 1
  • Gag VLPs group 2
  • the order of Env VLPs in the sequential immunizations (1. HIV-1 subtype C, 2. HIV-1 subtype B, 3. HIV-1 subtype D, 4. HIV-1 subtype A, and 5. HIV-1 subtype E), was determined by the phylogenetic homology of the HIV-1 Env proteins in between different HIV-1 subtypes.
  • VLPs containing 100 ⁇ g of Env and 25 ⁇ g of Gag proteins were administered at week 0, 4, 8, 12, and 16.
  • doses of VLPs were given through both i.m. and In. routes for comparison (groups 5 and 6, respectively) (Table
  • Immune serum and mucosal wash samples were collected for immune response assessments at 3 rd week after each vaccination. Blood samples were collected from the marginal ear vein of anesthetized animals for sera separation. Nasal and vaginal washes were collected by repeated flushing of the respective cavities with ice cold lavage medium (PBS,lx containing 150 mM PMSF, and 50 mM EDTA). PBMCs were isolated using Ficoll-paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA, USA) density gradient method as described earlier.
  • Env-specific antibody endpoint titers including serum IgG and its subtypes (IgGl, IgG2a, and IgG2b) and mucosal IgA, were determined by ELISA, using recombinant Con-S Env gpl20 protein (2 ⁇ g/ml) as the coating antigen.
  • the OD 450 was read with an ELISA reader (BioTek, Winooski, VT, USA) and data were compared with appropriate controls.
  • Antibody neutralization breadth and potency of immune serum and mucosal wash samples were evaluated using a highly sensitive, single-round pseudotype virus infectivity assay system.
  • a total of 32 HIV-1 Env-pseudotyped virions from tier 1, 2, and 3 of various isolates were generated for comparing the data of the neutralizing antibody assay, using the Fugene 6 transfection method (Promega, Madison, WI, USA) (Table 2).
  • Pseudoviruses were produced by co-transfection of 293T-cells with an Env-expressing plasmid of different subclades and Env- deficient HIV-1 genomic backbone plasmid, pSG3AEnv for TMZ-bl neutralization assays. These pseudoviruses exhibit a neutralization phenotype that is typical of most primary HIV-1 isolates. Neutralization activity was measured as the reduction of viral infectivity in comparison to that in control wells infected with virus alone.
  • ADCC was assessed by a standard 51 Cr release assay using 100 ⁇ (3.7 MBq) Na 51 Cr0 7 (Perkin Elmer, Downers Grove, IL, USA) for labeling.
  • Freshly isolated PBMCs from naive animals and 293 T-cells transfected with Env-expressing plasmid of different subclades were used as effector and target cells, respectively at an E:T ratio of 10: 1.
  • 51 Cr labelled target cells were incubated with 2-fold diluted serum samples and later co-cultured with effector cells.
  • 51 Cr activity was quantitated using liquid scintillation counter (Beckman Coulter, Atlanta, GA, USA) as described earlier 47 and the results were expressed as the percentage of lysis of target cells.
  • CFSE Carboxyfluorescein Diacetate Succinimidyl Ester
  • T-cell responses were carried out by the identification of degranulating CD8 + T-lymphocytes in vaccinated animals. Freshly isolated PBMCs from each group, were cultured and in vitro stimulated with appropriate antigens including negative and positive controls. After stimulation, PBMCs were stained with PE labelled anti-rabbit CD 107a antibody (0.5 mg/ml) along with monensin at the beginning of stimulation. At the end of the stimulation, cells were washed, and surface stained with APC-Cy7 labelled anti-rabbit CD8 antibody (0.5 mg/ml) (Biolegend, San Diego, CA, USA). The results were expressed as a percentage of CD8 + CD107a + T-cells and the data expressed was the one of the representative experiment.
  • Thl/Th2 cytokine e.g. IFN- ⁇ , IL-2, IL-6, and TNF-a
  • IL-2 IL-2
  • IL-6 IL-6
  • TNF-a TNF-a
  • BSA bovine serum albumin
  • Tests were performed using GraphPad Prism 7 software (San Diego, California), p values of O.05 (p ⁇ 0.05) were considered to be statistically significant. *p ⁇ 0.05; **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Example 2 Intranasal sequential immunization of HIV- 1 Env virus-like particles promotes mucosal TgA and high avidity effector CD8 T cell responses
  • Mucosal IgA antibodies are the first line of immune protection for many viral infections at mucosal surfaces, including HIV.
  • the mechanisms by which these antibodies can inhibit HIV-1 movement across the mucosal barrier include direct virus neutralization, viral aggregation, inhibition of transcytosis, intra-epithelial neutralization, phagocytosis, inhibition through mucus, and Fc receptor-mediated neutralization (antibody-dependent cellular cytotoxicity; ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Non-human primate (NHP) studies indicate that mucosal antibodies to gp41 have important functional roles in protection for HIV infection. Thus, there is a resurgence of interest in the role of mucosal IgA antibodies in protection against HIV-1.
  • HIV-specific T cell responses confer protection against the clinical progression of disease after vims infection and help in controlling, if not clearing, HIV infection .
  • the role of CD4 T cells is a major part of the induction and maintenance of memory CD8 T cell and B cell responses. HIV-specific CD4 T cell responses play an imperative role in controlling viral replication during infection. Even more strikingly than CD4 T cells, HIV-specific CD8 T cell or cytotoxic T lymphocyte (CTL) responses also play a key role in controlling viral replication.
  • CTL cytotoxic T lymphocyte
  • VLPs HIV-1 Env viruslike particles
  • Other studies have attempted to elicit mucosal immunity against HIV using heterologous prime-boost immunizations, but none have attempted to simulate HIV evolution/escape through a panel of "native-like" Env VLPs.
  • HIV-specific mucosal antibody and T cell immune responses were induced in rabbits with intranasal (i.n.) sequential immunizations of a panel of Env-enriched VLPs from HIV-1 clades A-E.
  • the sequential immunization regimen enhanced the CD3 + CD4 + T cell population compared with the Gag or single Env VLPs- vaccinated groups of animals ( Figure 10A). Also, sequentially immunized animals had increased CD3 + CD8 + T cell proliferation when compared with the Gag or single Env VLPs-vaccinated animals ( Figure 10B). Thus, sequentially immunized animals increased both the CD4 and CD8 T cell proliferation when compared with the other animal groups.
  • TNF-a levels were significantly (p ⁇ 0.01) increased in the sequentially immunized group compared with the other groups ( Figure 11C).
  • IL-6 levels were undetectable in all the groups ( Figure 1 ID). Cytokine ELISA results showed that sequential immunizations induced Thl -type cytokines in the circulation.
  • the sequential immunization group also showed a significantly (p ⁇ 0.05) higher number (5-10-fold) of IFN-y-producing cells than the Gag VLPs or single Env VLPs-immunized groups ( Figure 1 IE).
  • the IFN- ⁇ ELISPOT data were comparable with the IFN- ⁇ ELISA results.
  • the sequential immunization group had a significantly (p ⁇ 0.01) higher percentage of CD8 + IFN-y-producing population compared with other control groups.
  • the Gag VLPs group did not show any increase in the CD8 + IFN-y-producing cell percentage, but the single Env VLPs group had a modest level of enhancement in this percentage.
  • FACS data also revealed that sequential immunization group had an increase in CD4 + IFN-y-producing population compared with other control groups ( Figure 1 IF).
  • the levels of degranulation was determined by analyzing the frequencies of double positive CD4 + CD107a + and
  • CD8 + CD 107a + T-subsets A slight increase in the percentage of CD4 + CD 107a + cells were observed in the sequentially immunized group versus other vaccination groups ( Figure 12A). The CD8 + CD107a + T cells in the sequential immunization group of animals were increased when compared with the other groups ( Figure 12B). These data support that sequential immunizations with various Env VLPs induced CD8 T cell cytolytic immune responses.
  • CD8 TCR avidity was tested using a FACS method after gating CD8 T cell populations with the IFN- ⁇ expression at different antigen concentrations. Sequentially immunized animals showed an increase in the TCR avidities compared with the repetitive-immunizations of Gag only or single Env VLPs ( Figure 13C). The increases induced by sequential immunization regimen in CD8 TCR avidities and cytokine polyfunctionality underlies the improved antiviral immune responses of the group.
  • IgA levels at local and distal mucosal compartments IgG2- dominant serum antibody titers
  • T cell proliferation T cell proliferation
  • Thl cytokine production especially IFN- ⁇
  • cytokine polyfunctionality high avidity CD8 T cell responses.
  • the sequential immunization group significantly enhanced the mucosal IgA levels in nasal and vaginal secretions. Given that the majority of HIV transmission occurs via mucosal surfaces, there is a great interest in strengthening the innate and adaptive mucosal environment which can shape the outcome of HIV infection.
  • Anti-HIV-1 mucosal IgA has not been observed only in HIV-1 infected individuals, but also in some HIV-exposed seronegative individuals (HESNs). IgA isolated from cervicovaginal secretions of these individuals are capable of inhibiting viral infection. Because of mucosal IgA's importance in HIV prevention, vaccine procedures capable of eliciting a strong mucosal IgA response are beneficial by contributing to the containment of HIV-1 infection.
  • Sequential immunizations enhanced antigen-specific T lymphocyte proliferation. As shown herein sequential immunization regimen generated a broad-spectrum T cell-based immunity with enhanced T lymphocyte proliferation. Because circulating primed T cells have a lower activation threshold than naive T cells, they preferentially activate when presented with conserved epitopes during the sequential immunization with Env VLPs. The finding supports that sequential immunizations can be designed to stimulate cellular immune responses.
  • the sequential immunization group exhibited a Thl type immune responses, demonstrated by the IgG2-dominant antibody responses and by high levels of Thl cytokines (IFN- ⁇ , IL-2, and TNF-a) in PBMCs' culture.
  • the results of ELISPOT and ICS demonstrate that the animals vaccinated through sequential immunizations had enhanced IFN- ⁇ production, especially by CD8 T cells.
  • Preliminary evidence and different NHP studies have shown a vigorous T cell response (including IFN- ⁇ production) is an immune correlate of protection against HIV-1/SIV infection.
  • the sequential immunizations with various Env VLPs induced CD8 T cell cytolytic immune responses.
  • a crucial role of CD8 T lymphocytes in protection from intracellular pathogens such as viruses like HIV-1 supports the results.
  • CD8 functionalities are the key determinant in antiviral immunity. More recently, studies have shown that the exclusive determinant of HIV viremic control in progressors was the amount of killing of HIV-infected targets by high avidity CD8 T cells indicating that CD8 T cell avidity with cytokine polyfunctionality can underlie HIV control.
  • IFN- ⁇ affects a wide range of target cells and induces the host defense against infectious agents by up-regulating MHC class I and II proteins on a variety of cells, like macrophages and epithelial cells; acting on CD4 Thl differentiation; regulating the production of a variety of other pro -inflammatory cytokines, including IL-2 and TNF-a; and stabilizing inflammatory T cell responses.
  • heterologous prime/boost vaccination strategy with other vaccine vectors or as a standalone vaccine approach can significantly improve HIV vaccines.
  • SF9 Spodoptera frugiperda
  • ATCC HEK293T cells
  • DMEM Dulbecco's Modified Eagle's Medium
  • the NIH AIDS Research and Reference Reagent Program provided the HIV-1 Env clones of various strains, soluble human CD4, HIV-1 SF162 Env, Gag recombinant protein pr55, HIV Con-S Env protein and peptide pool, goat anti-HIV-1 Env polyclonal antibody, and monoclonal antibodies (e.g. PGT126, PGT128, and PGT145).
  • rBVs bacuioviruses
  • clades A-E various HIV- 1 Env
  • Modified Env gene constructs were generated by replacing the original signal peptide encoding sequences with the honeybee melittin sequence and adding a trimeric form of leucine zipper sequence (GCN4pii) to the C-termini of Env cytoplasmic sequences to increase Env glycoprotein production in insect cells and to express conformation-stabilized trimeric Env proteins, respectively.
  • GCN4pii trimeric form of leucine zipper sequence
  • These Env were from strains; 92UG037.8 (A), SF162 (B), ZM53M.PB12 (C), 92UG021.16 (D), and 93TH976.17 (E).
  • Env based was chosen on the similarity in their amino acid sequences and phylogenetic homology of the HIV-1 Env proteins between different HIV-1 subtypes.
  • HIV-1 Env (Env/Gag) VLPs were produced by co-infection of SF9 cells with rBVs expressing trimeric Env and Gag at the multiplicity of infections (MOIs) of 2:1.
  • MOIs multiplicity of infections
  • Gag VLPs produced by infection of SF9 cells with rBVs expressing Gag alone were used as a control group.
  • VLPs were concentrated by porous fiber filtration using a AKTA Flux (GE Healthcare) and further purified using a sucrose density gradient centrifugation method.
  • immunization regimen was determined based on the ability to maximize genetic divergence, which can increase the possibility of generating broader immune responses.
  • Five doses of VLPs containing lOC ⁇ g of Env or/and 25 ⁇ g of Gag proteins in total per rabbit were administered through i.n. route.
  • Blood and mucosal lavages were collected at one- week pre- (preimmune) and 3 -week after the last vaccination.
  • Blood samples were collected from the marginal ear vein of anesthetized animals using vacuette blood collection tubes containing sodium heparin (Greiner Bio-One) for peripheral blood mononuclear cells (PBMCs) and sera separation.
  • Sera were isolated from the clotted blood and PBMCs were harvested using the Ficoll Paque PLUS (GE Healthcare) density gradient method (Miltenyi Biotec). Sterile cotton tipped swabs were used to collect nasal/vaginal cavity material.
  • Each individual swab was placed into ice-cold lavage buffer (0.9%,w/v, NaCl; 0.05%,v/v, Tween 20; 0.1%,w/v, NaN 3 ; and Imol/dm 3 PMSF).
  • the swabs were vortexed in the buffer and all collection tubes per rabbit were pooled, mixed, aliquoted, and stored at -80°C.
  • Serum (IgG, IgGl, IgG2a, and IgG2b) and mucosal IgA (nasal and vaginal) levels were determined against Con-S Env (consensus) and various Env (autologous) antigens using indirect ELISA and cell-based ELISA, respectively.
  • Env autologous antigens
  • cell-based ELISA HEK293T cells were transfected with various HIV-1 Env clones (the same HIV-1 Env plasmids that were used in the Env VLPs), influenza full-length (FL) HA, or Gag (non-Env) with Lipofectamine 2000 (Invitrogen).
  • Influenza FL HA and Gag transfected cells were used as control groups in the assay.
  • the HIV Con-S Env protein (200ng/well) and the fixed transfected HEK293T cells (5 xlO 4 cells/well) were used as coating antigens and the color was developed as described earlier using anti-rabbit HRP-conjugated IgG/IgA antibodies (ThermoFisher).
  • the optical density at 450nm (OD 45 o) was read with an ELISA reader (BioTek).
  • HIV-1 pseudoviruses (7) HIV-1 pseudoviruses.
  • the avidity indices of systemic and mucosal antibodies to Env proteins were determined by ELISA in the presence of 1.5M sodium thiocyanate (NaSCN). Pseudotype virions purified by filtration, pelleted, and disrupted with 1% Triton X-100 were used as coating antigens.
  • the TZM-bl neutralization assay was used with minor modifications. Two-fold serial dilutions of sera/mucosal wash samples from individual animals were plated and
  • CD3 + CD4 + and CD3 + CD8 + T cell proliferation were measured by a FACS method using PBMCs, harvested from the vaccinated animals.
  • PBMCs were in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone.
  • Stimulated PBMCs were stained with anti-rabbit CD3 antibody (Biorad), coupled with anti-rabbit IgG-Pacific-blue secondary antibody (Thermofisher). After washing, these cells were stained for anti-rabbit CD4 or CD8ct antibodies (Biorad), coupled with anti-rabbit IgG-APC or FITC secondary antibodies (Abeam), respectively.
  • CD3 + CD47CD8 + T cell proliferation was analyzed with a BD FACSCanto II flow cytometer (BD Biosciences) using FlowJo Software (Treestar Inc).
  • Thl/Th2 cytokines IFN- ⁇ , IL-2, TNF-a, and IL-6
  • PBMCs were in vitro stimulated with HIV-1 Con-S Env peptides, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone.
  • Cytokine concentrations were calculated from their respective standard curves and data were represented in pg/ml for each group of rabbits from three independent experiments.
  • IFN- ⁇ ELISPOT assays were performed. 96-well filtration plates (Millipore) were coated with 2 ⁇ g/ml of purified anti-rabbit IFN- ⁇ antibody (Mabtech). After washing and blocking, freshly prepared PBMCs (l xlO 6 cells/well) were incubated in triplicate with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone for 18- 20h. Plates were washed, incubated with biotinylated anti-rabbit IFN- ⁇ antibody (Mabtech) and further with anti-rabbit streptavidin-HRP IgG secondary antibody (Jackson ImmunoResearch). Spots were developed using 3,3'-diaminobenzidine (DAB) (Pierce) and counted by an ELISPOT reader (Biosys).
  • DAB 3,3'-diaminobenzidine
  • PBMCs were cultured and in vitro stimulated with HIV-1 Con-S Env peptide pool, PMA (50ng/ml) plus ionomycin (500ng/ml), or media alone.
  • PBMCs were stained with anti-rabbit CD 107a antibody (Abeam), coupled with anti -rabbit IgG-PE secondary antibody (Abeam) along with monensin (BD Biosciences).
  • Antigen-specific cytokine-producing cells were quantified by an intracellular cytokine staining (ICS) assay.
  • PBMCs l xlO 6 cells/well
  • PMA 50ng/ml
  • ionomycin 500ng/ml
  • media alone in the presence of 5 ⁇ /ml of brefeldin A (Golgi Plug) (BD Biosciences).
  • BD Biosciences following washing and blocking, cells were surface stained with anti-rabbit CD4 and CD8 antibodies (Biorad), coupled with Per-CP and APC-Cy7 conjugated secondary antibodies, respectively.
  • CD4/CD8 T cells were gated with IFN- ⁇ expression at specific antigen concentration for measuring IFN- ⁇ producing CD4 or CD8 T cells.
  • CD4 or CD8 T cell populations were gated and single, double or triple cytokine expressing cell percentages were enumerated. The percentage cytokine expression of each animal was averaged within the group and is expressed as a pie-chart of relative
  • CD8 T cell receptor (TCR) avidity CD8 T cells were gated with IFN- ⁇ expression and was measured as percent (%) maximum response at different antigen concentrations. (15) Statistical analysis.
  • Nonlinear regression plots depict the regression results (line) with 95% confidence intervals (cloud) computed from results calculated from multiple independent experiments. Levels of significance (p-value) were compared between sequential immunization group and other control groups. Tests were performed using Graph Pad Prism 7 software, p- values of ⁇ 0.05 (p ⁇ 0.05) were considered to be statistically significant.
  • CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature, 2003. 421(6925): p. 852-6.
  • Mohan, T., et al. Sequential immunizations with a panel of HIV-1 Env virus-like particles coach immune system to make broadly neutralizing antibodies. Sci Rep, 2018. 8(1): p. 7807.
  • Mohan, T., Mitra, D. & Rao, D.N. Nasal delivery of PLG microparticle encapsulated defensin peptides adjuvanted gp41 antigen confers strong and long-lasting immunoprotective response against HIV-1. Immunol Res 58, 139-153 (2014).
  • Subbarao B. et al. Lyb-7, a new B cell alloantigen controlled by genes linked to the IgCH locus.

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Abstract

L'invention concerne une nouvelle stratégie d'immunisation séquentielle et des trousses permettant sa mise en œuvre.
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