WO2002005846A1

WO2002005846A1 - Quadrivalent combination vaccine including diphtheria toxoid, tetanus toxoid, whole cell pertussis and hepatitis b surface antigen, and preparation method thereof

Info

Publication number: WO2002005846A1
Application number: PCT/KR2001/001153
Authority: WO
Inventors: Cheon-Soon Bae; Gwan-Yeul Lim; Kyung-Nam Park; Hong-Joo Kim; Dal-Ho Um; Jong-Soo Kim
Original assignee: Green Cross Vaccine Corp
Current assignee: Green Cross Vaccine Corp
Priority date: 2000-07-05
Filing date: 2001-07-05
Publication date: 2002-01-24
Anticipated expiration: 2003-01-05
Also published as: CN1383384A; CN1211121C; KR100385711B1; KR20020005081A; AU2001269569A1

Abstract

A quadrivalent (DTwPH) combination vaccine comprising diphtheria toxoid, tetanus toxoid, whole cell pertussis, and HBsAg, and a method for preparing the same are provided. In the preparation of the DTwPH combination vaccine, diphtheria toxoid and tetanus toxoid are adsorbed onto aluminum phosphate (AlPO4) gels, and HBsAg is adsorbed onto aluminum hydroxide (Al(OH)3) gel. The DTwPH combination vaccine is adjusted to have a final pH of 6.5-7.5, and the concentrations of constituents also adjusted with the concentration of aluminum hydroxide gel in the range of 15-35 νgAl/mL.

Description

QUADRIVALENT COMBINATION VACCINE INCLUDING DIPHTHERIA

TOXOID, TETANUS TOXOID, WHOLE CELL PERTUSSIS AND

HEPATITIS B SURFACE ANTIGEN AND PREPARATION METHOD

THEREOF

FIELD OF THE INVENTION

The present invention relates to a quadrivalent combination vaccine

(hereinafter, DTwPH combination vaccine) including diphtheria toxoid, tetanus

toxoid, whole cell pertussis, and Hepatitis B surface antigen (hereinafter, HBsAg)

and a method for preparing the same.

Included in the Expanded Program on Immunization (EPI) by the World

Health Organization (WHO), DTP combination vaccines for simultaneous

vaccination against diphtheria, tetanus, and pertussis have been recommended as

essential vaccines for infants in almost all worldwide countries. Hepatitis B

infections, which are mostly common in Southeast Asia, Central Asia, Africa, and

South America East South Asia, are known as a potential cause of hepatocarcinoma.

However, Hepatitis B infections also can be prevented through immunization so that

world-wide vaccination activities with Hepatitis B vaccine have been actively

conducted by WHO. Recently, to eliminate the inconvenience of separate vaccinations with DTP

combination vaccine and Hepatitis B vaccine that have different administration

schedules, and to supply these vaccines to underdeveloped countries at reduced costs

to increase their vaccination ratio, a combination vaccine product (DTaPH) including

diphtheria toxoid, tetanus toxoid, acellular pertussis and HBsAg has become

commercially available (European Patent Publication No. 0 642 355 Bl, July 15,

1998; Korean Patent Publication No. 1992-0009729, Green Cross Corp.).

BACKGROUND OF THE INVENTION

In association with the preparation of a DTwPH combination vaccine, active

research has been conducted to solve problems encountered in pharmaceutical

preparation of the combination vaccine to prevent toxicity increase while keeping the

immunogenicity of HBsAg.

The problems occurring in the pharmaceutical preparation of the

combination vaccine include aggregation of individual vaccine solutions and vaccine

adhesion to glass storage bottle. Here, the term "aggregation" refers to the

formation of solid substance that is not suspended by gentle hand shaking, and the

term "adhesion" refers to the binding of DTwPH combination vaccine particles to the

wall of a storage glass bottle, which is strong enough not to be separated by gentle

shaking. The aggregation in the DTwPH combination vaccine occurs by electrostatic attraction between individual vaccine adsorbed solutions and pertussis bacteria and,

especially between aluminum hydroxide gel used as an adsorbing agent and pertussis

bacteria. This adhesion occurs by long-time reaction between DTwPH combination

vaccine particles and glass wall. As the DTwPH combination vaccine is stored for a

long period of time, stability of the combination vaccine product seriously degrades

due to the aggregation and adhesion phenomena.

The DTwPH combination vaccine has a problem of increasing toxicity due

to the excess use of adsorbing agent and protein. Besides, since HBsAg has a low

incompatibility with other antigens, its immunogenicity therein is decreased

compared to simultaneous administration of two separate vaccines, DPT combination

vaccine and Hepatitis B vaccine (WHO headquarters, Informal consultation on

quardrivalent dipheria-tetanus-pertussis-hepatitis B vaccine, 1992).

Therefore, it is an object of the present invention to prevent effectively the

said "aggregation" and adhesion" phenomena, and also to provide a new DTwPH

combination vaccine and a preparation method thereof which can maintain the

immnogenicity of HBsAg with the same geometric mean of antibody titre (ab GMT)

and the formation ratio of effective antibody titre as in a simultaneous administration

with separate trivalent DTP combination vaccine and single antigen Hepatitis B

vaccine. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new quadrivalent DTwPH combination

vaccine, including diphtheria toxoid, tetanus toxoid, whole cell pertussis, and HBsAg,

in which diphtheria toxoid and tetanus toxoid are adsorbed onto aluminum phosphate

(AlPO₄) gels and HBsAg is adsorbed onto aluminum hydroxide (Al(OH) ) gel.

The present invention also provides a preparation method of DTwPH

combination vaccine, comprising (a) adjusting pH of diphtheria toxoid adsorbed

aluminum phosphate gel solution, tetanus toxoid adsorbed aluminum phosphate gel

solution, and HBsAg adsorbed aluminum hydroxide gel solution and a pertussis

stock solution in the range of 6.5-7.5 before preparation of the final DTwPH

combination vaccine composition; (b) mixing the pH-adjusted diphtheria toxoid and

tetanus toxoid adsored solutions; and (c) mixing the mixture obtained in step (b) with

the pH-adjusted HBsAg adsored solution and the inactivated pertussis stock solution.

The present invention also provides a combination vaccine storage method,

comprising adding gelatin to a common combination vaccine or the DTwPH

combination vaccine described above, or using a silicon-coated glass bottle as a

storage bottle for the combination vaccine. It is preferable that the concentration of

the gelatin is in the range of 0.1-0.7 w/v%.

Hereinafter, the structure and effect of the present invention will be described

in greater detail. A diphtheria toxoid stock solution used in the present invention is prepared

by culturing Corynebacterium diphtheriae Park Willium strain. The resulting

culture supernatent is filtered, precipitated, and purified after detoxification with

formalin to obtain a suitable diphtheria toxoid stock solution. Suitable diphtheria

toxoid stock solutions can be obtained by using strains or methods other than

described above.

A tetanus toxoid stock solution is prepared by culturing Clostridium tetani

Harvard strain. The resulting culture solution is filtered, precipitated, and purified

after detoxification with formalin to obtain a suitable diphtheria toxoid stock solution.

Suitable diphtheria toxoid stock solutions can be obtained by using strains or

methods other than described above.

A pertussis stock solution is prepared by culturing Bordetella pertussis. The

resulting culture solution is centrifuged. The obtained pertussis pellet is suspended

in saline and inactivated by treatment to obtain a suitable inactivated pertussis stock

solution. Suitable pertussis stock solutions can be obtained by using strains or

methods other than described above.

HBsAg is extracted through homogenization from a recombinant yeast

culture. The HbsAg from recombinant yeast is subjected to a series of separation

methods including ion chromatography, ultrafiltration, dialysis, ultracentrifugation,

gel filtration chromatography, etc. to obtain a suitable HBsAg stock solution. Suitable HBsAgs can be obtained by using methods other than described above and

HbsAgs from plasma can be also used.

Suitable adsorbing agents used in the present invention include aluminum

hydroxide and aluminum phosphate gels. Aluminum hydroxide and aluminum

phosphate gels can be purchased or prepared by known methods.

A silicon-coated bottle used for storage of combination vaccines in the

present invention is manufactured by soaking a glass bottle in a 1 :40 dilute solution

of 35% polymethylsiloxane (Dow Corning 365 silicon emulsion) and drying with

heat the glass bottle at 310° C for 10 minutes. Any silicon-coated glass bottles

manufactured by methods other than described above and commercially available

silicon-coated bottles can be used.

The DTwPH combination vaccine according to the present invention is

prepared by separately adsorbing diphtheria toxoid, tetanus toxoid, and HBsAg stock

solutions on to aluminum gels, and mixing the above solutions with a pertussis stock

solution, adjusting each single antigen vaccine concentrations to appropriate level.

The diphtheria toxoid stock and tetanus toxoid stock solutions are adsorbed onto

aluminum phosphate gels, and the HBsAg stock solution is adsorbed onto aluminum

hydroxide gel.

The single antigen vaccine concentrations in the DTwPH combination

vaccine according to the present invention are 20-50 Lf/mL for diphtheria toxoid, 4- 20 Lf/mL for tetanus toxoid, 10-24 OU/mL for pertussis, and 10-30 μg/rήL for

HBsAg. The concentration of aluminum hydroxide gel is in the range of 15-35

gAl/mL. Total aluminum gel concentration is in the range of 550-1000 /zgAl/mL.

The terms "the concentration of aluminum hydroxide gel" or "aluminum gel

concentration" mean the concentration of Al in aluminum gel.

FIG. 1 is a lOOX-magnified photograph showing the degree of aggregation of

pertussis bacteria with aluminum hydroxide gel with respect to the concentration of

aluminum hydroxide gel. When aluminum hydroxide gel and pertussis bacteria

are mixed, aggregation occurs by electrostatic attraction. To minimize the

aggregation between aluminum hydroxide gel and pertussis, the determination of the

concentration of aluminum hydroxide gel is considered to be a significant factor.

When the final concentration of pertussis bacteria is 24 OU/mL, no aggregation is

observed at the concentration of aluminum hydroxide gel of 35 βgAVmL, as shown

in FIG. 1(a). Aluminum hydroxide gel and pertussis bacteria start to aggregate at

the concentration of aluminum hydroxide gel no less than 35 gAl/mL, and the

degree of the aggregation becomes serious at the concentration of aluminum

hydroxide gel no less than 200 jUgAl/mL, as shown in FIG. 1(b).

Therefore, the concentration of aluminum hydroxide gel onto which HBsAg

is adsorbed is preferably in the range of 15-35 / gAl/mL based on the final

combination vaccine composition. The diphtheria toxoid adsorbed solution has zero charge or negative charges

at neutral pH and positive charges at weak acidic pH. Meanwhile, pertussis bacteria

adsorbed solution have negative surface charges at neutral pH. Therefore, when the

pertussius stock solution and the diphtheria toxoid adsorbed solution are mixed after

pH adjustment in the range of 6.5-7.5 (measured by a pH meter, Zeta-Meter System

3.0, NJB PACIFIC INC., USA), aggregation in the mixed solution can be prevented

by electrostatic repulsive force.

When adjusting each pH of diphtheria toxoid, tetanus toxoid, and HBsAg

adsorbed solutions and the pertussis stock solution in the range of 6.5-7.5 and mixing

them together, the final DTwPH combination vaccine composition has a pH of 6.5-

7.5 so that aggregation between single antigen vaccines can be prevented.

The DTwPH combination vaccine according to the present invention prepared

by the method described above meets the requirement of biological preparation

standards (by Korea Food and Drug Administration, 1999) for the respective single

antigen vaccines with reduced toxicity. In addition, the immunogenicity of HBsAg

is stable in the DTwPH combination vaccine. Here, the stability of immunogenicity

means that the immunogenicity of HBsAg is maintained at or slightly increased from

an initial level without significant reduction after 3-month storage at 4° C.

Undesirable aggregation or adhesion of vaccines to the wall of a glass bottle

during storage can be prevented by addition of a high-viscosity material such as gelatin in an amount of 0.1-0.7 w/v% to common combination vaccines or to the

DTwPH combination vaccine according to the present invention and by use of a

silicon-coated glass bottle for storage. As a result, common combination vaccines

or the DTwPH combination vaccine according to the present invention are kept in a

white stable suspension during storage. Here, the storage stability means that the

combination vaccine is kept in a white suspension without vaccine aggregation and

adhesion to storage bottle after 3-month storage in a cold chamber at a temperature

of2-8° C.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a lOOX-magnified photograph showing the degree of aggregation

of pertussis bacteria with aluminum hydroxide gel with respect to the concentration

of aluminum hydroxide gel.

FIG. 2 is a Western blot photograph showing the adsorption degree of

diphtheria toxoid adsorbed solution.

FIG. 3 is a lOOX-magnified photograph taken by an optical microscope and

showing the effect of use of silicon-coated glass bottle for storage on the degree of

aggregation in DTwPH combination vaccine. EXAMPLES

The present invention will be described in greater detail by means of the

following examples. The following examples are for illustrative purposes and are

not intended to limit the scope of the invention.

Example 1: Preparation of Diphtheria Toxoid Stock Solution

A diphtheria toxoid stock solution used in the present invention was prepared

by culturing Corynebacterium diphtheriae Park Willium strain. Formalin at a final

concentration of 6% was added to the resulting culture supernatent and left at 36° C

for 28 days for detoxification, followed by purification through ultrafiltration,

dialysis, precipitation with ammonium sulfate, gel filtration chromatography, and

then dialysis to obtain a diphtheria toxoid stock solution.

Example 2: Preparation of Tetanus Toxoid Stock Solution

A tetanus toxoid stock solution was prepared by culturing Clostridium tetani

Harvard strain. Formalin at a final concentration of 0.45% was added to the

resulting culture solution and left at 36° C for 28 days for detoxification, followed

by purification by microfiltration, ultrafiltration, dialysis, precipitation with

ammonium sulfate, gel filtration chromatography, and then dialysis to obtain a

tetanus toxoid stock solution. Example 3: Preparation of Whole Cell Pertussis Stock Solution

A pertussis stock solution was prepared by culturing Bordetella pertussis.

The resulting culture solution was centrifuged. The obtained pertussis pellet was

suspended in saline and inactivated by treatment at 56° C for 30 minutes to obtain a

suitable pertussis stock solution.

Example 4: Preparation of HBsAg Stock Solution

HBsAg was prepared by culturing recombinant Hansenula polymorpha.

The resultant culture was subjected to homogenization for cell extraction and then

ion chromatography, ultrafiltration, dialysis, ultracentrifugation, and gel filtration

chromatography to obtain a suitable HBsAg stock solution.

Example 5: Preparation of Diphtheria Toxoid and Tetanus Toxoid Adsorbed

Solutions

Diphtheria toxoid and tetanus toxoid stock solutions were adsorbed onto

aluminum phosphate gels (pH 5.5) by mixing with stirring and diluted with isotonic

saline if necessary. Final protein and toxoid concentrations in the adsorbed solution

can be appropriately adjusted. In this example, the concentration of diphtheria

toxoid solution was adjusted to be about 280 Lf/mL, and the concentration of tetanus toxoid solution was adjusted to be about 50 Lf/ml. The respective adsorbed

solutions had a pH of 5.5-6.0. The adsorption of diphtheria toxoid onto aluminum

phosphate gel was observed by high-performance liquid chromatography (HPLC)

and a Western Blot analysis. The result of the Western blot analysis is shown in

FIG. 2. In FIG. 2, Lane 1 is for a molecular weight marker, Lanes 2, 3, and 4 for

diphtheria solutions of 0.025 Lf/mL, 0.05 Lf/mL, and 0.10 Lf/mL, respectively, and

Lane 5 for supernatant of 0.4 Lf/mL from centrifugation of the diphtheria adsorbed

solution (equivalent to 100% desorption of diphtheria toxoid). The prepared

diphtheria toxoid and tetanus toxoid adsorbed solutions were stored in a cold

chamber at a temperature of 2-8° C.

Example 6: Preparation of HBsAg Adsorbed Solution

To enhance immunogenicity, HBsAg was adsorbed onto aluminum hydroxide

gel. The HBsAg stock solution and aluminum hydroxide gel were mixed with

stirring to adsorb the HBsAg stock solution onto the gel and diluted with isotonic

saline if necessary. The concentration of aluminum hydroxide gel was adjusted to

be 60 jUgAl/mL, and the concentration of HBsAg stock solution was adjusted to be

72 jUg/mL. The adsorption of HBsAg onto aluminum hydroxide gel was observed

by HPLC and a Western Blot analysis. The prepared HBsAg adsorption solution

was stored in a cold chamber at a temperature of 2-8° C. Example 7: Preparation of DTwPH Combination Vaccine

The adsorbed solutions prepared in Examples 5 and 6 and the pertussis stock

solution (192 OU/mL) were mixed to obtain the final DTwPH combination vaccine

composition. To prevent aggregation in the mixture of the pertussis stock solution

and the adsorbed solutions by electrostatic attraction, the respective solutions were

pH adjusted at 7.1 before mixing.

First, the diphtheria toxoid adsorbed solution was mixed with the tetanus

toxoid adsorbed solution, and the HBsAg adsorbed solution and an inactivated

pertussis stock solution were added to the mixture to prepare the DTwPH

combination vaccine composition. Next, aluminum phosphate gel was added to the

DTwPH combination vaccine composition to adjust total aluminum gel concentration

to a desired level. The concentration of the respective stock solutions can be

adjusted by addition of saline or phosphate buffer solution. Thimerosal was added

as an antiseptic in an amount of 0.05-0.1 mg/mL. 2-Phenoxyethanol can be added in

an amount of 3-6 mg/mL if necessary. The final DTwPH combination vaccine

composition had the composition shown in Table 1 at pH of 7.1.

Table 1

Experimental Example 1: Immunogenicity Test for HBsAg in DTwPH

Combination Vaccine

To determine the immunogenicity of HBsAg in the DTwPH combination

vaccine prepared in Example 7, four to five weak-old, thirty ICR mice were

immunized subcutaneously at the abdomen with the combination vaccine. After 4

weeks, blood was withdrawn, followed by determination of the geometric mean of

antibody titre (Ab GMT) and the percentage of mice having an effective antibody

titre for the immunogenicity test. Here, Ab GMT means the geometric mean of

mice antibody titres no less than 1 mlU/mL, and the percentage of mice having an

effective antibody titre means the percentage of mice having an antibody titre no less

than 10 mlU/mL in the all experimental mice.

As a control group for simultaneous administration, mice were immunized

subcutaneously at different sites with a 0.5-mL trivalent DTwP combination vaccine

having the same antigen composition as in Example 1 (40 Lf/mL diphtheria toxoid,

15 Lf/mL tetanus toxoid, and 24 OU/mL pertussis bacteria) and a 0.5-mL single antigen Hepatitis B vaccine (24 /g/mL), followed by the immunigenicity test for

HBsAg by the same method described above.

As shown in Table 2, the DTwPH combination vaccine shows a stable

immunogenicity for HBsAg (in Ab GMT and mice percentage having effective

antibody titre) as in the simultaneous administration of separate vaccines.

Table 2

Experimental Example 2: Potency Test for DTwPH Combination Vaccine

Potency test for the DTwPH combination vaccine prepared in Example 7 of

respective diphtheria toxoid, tetanus toxoid, whole cell pertussis, and HBsAg was

performed according to the potency requirement of biological medicine by Korea

Food and Drug Administration (1999, 122-130 diphtheria, tetanus, pertussis

adsorbed combination vaccine; and 310-313 Hepatitis B vaccine).

The results were 2-4 units/mL of diphtheria toxoid, 4 units/mL or greater

against tetanus toxoid, and 10 IU/mL against whole cell pertussis. The potency against HBsAg was equivalent to or greater than a reference (used as a standard in

Green Cross Corporation). Evidently, the DTwPH combination vaccine satisfies the

potency requirement of biological medicine.

Example 8: Use of Gelatin

To increase the viscosity of the DTwPH combination vaccine composition

prepared in Example 7, gelatin was added at a final concentration of 0.6 w/v%. As

a result, no vaccine aggregation and adhesion to glass wall was observed after 3-

months storage at 2-8° C.

Example 9: Use of Silicon-coated Glass Bottle

0.5 ml of the DTwPH combination vaccine composition prepared in

Example 7 was portioned into a silicon-coated glass bottle (3mL) and a non-coated

glass bottle and stored for 3 months or longer at 2-8° C. As a result, vaccine

adhesion to the wall of the silicon-coated glass bottle was not observed. After

shaking the two bottles vigorously, the phase stability of the vaccine composition

was observed using a microscope at 100X (Axioplan, Carl Zeiss Co., Germany).

For the DTwPH combination vaccine composition stored in the silicon-coated glass

bottle, the initial uniform dispersion was maintained during storage, as shown in FIG.

3(a). In contrast, aggregation of DTwPH combination vaccine particles was observed in the non-silicon coated glass bottle, as shown in FIG. 3(b).

INDUSTRIAL APPLICABILITY

As described above, vaccine aggregation and adhesion to storage glass bottle

can be prevented by preparing the DTwPH combination vaccine composition after

adjusting the concentration of aluminum hydroxide gel and the pH of the respective

adsorbed solutions (diphtheria toxoid adsorbed solution, tetanus toxoid adsorbed

solution, and HBsAg adsorbed solution) and pertussis stock solution, or by adding

gelatin to the DTwPH combination vaccine composition, or by using a silicon-coated

glass bottle for storage.

The immunogenicity of HBsAg can be maintained in the DTwPH

combination vaccine and the potencies of the respective components can be satisfied

with the requirement of biological preparation standards (by Korea Food and Drug

Administration, 1999), by preparing the DTwPH combination vaccine after

adsorbing diphtheria toxoid and tetanus toxoid onto aluminum phosphate gels and

HBsAg onto aluminum hydroxide gel, and adjusting the concentrations of each

component.

In addition, other single antigen vaccines such as Heamophilus influenzae

type b (Hib) vaccine or inactivated Polio vaccine can be added in the preparation of

the DTwPH combination vaccine or can be mixed with the same immediately before injection for simultaneous immunization.

Claims

What is claimed is:

1. A DTwPH (Diphtheria-tetanus-pertusis-hepatitis) combination

vaccine in which diphtheria toxoid and tetanus toxoid are adsorbed onto aluminum

phosphate gels, and Hepatitis B surface antigen is adsorbed onto aluminum

hydroxide gel.

2. The DTwPH combination vaccine of claim 1, having a final pH of

6.5-7.5.

3. The DTwPH combination vaccine of any of claims 1 and 2, wherein

the concentration of the aluminum hydroxide gel is in the range of 15-35 g Al/mL.

4. The DTwPH combination vaccine of any of claims 1 through 3,

wherein the concentration of diphtheria toxoid is in the range of 20-50 Lf/mL, the

concentration of tetanus toxoid is in the range of 4-20 Lf/mL, the concentration of

pertussis is in the range of 10-24 OU/mL, the concentration of Hepatitis B surface

antigen is in the range of 10-30 βg/mL, and total aluminum gel concentration is in

the range of 550-1000 /zgAl/mL.

5. A combination vaccine, which comprises gelatin in an amount of

0.1-0.7w/v%.

6. The combination vaccine of claim 5, wherein the amount of gelatin

is in the range of 0.5-0.7 w/v%.

7. The combination vaccine of any of claims 5 and 6, wherein the

combination vaccine is a DTwPH combination vaccine which is used against

diphtheria, tetanus, pertussis, and hepatitis type B.

8. A method for preparing a DTwPH (diphtheria-tetanus-pertusis-

hepatitis) combination vaccine, the method comprising:

mixing a diphtheria toxoid adsorbed aluminum phosphate gel solution and a

tetanus toxoid adsorbed aluminum phosphate gel solution (a); and

mixing the mixture obtained in step (a) with a Hepatitis B surface antigen

adsorbed aluminum hydroxide gel solution and an inactivated pertussis stock

solution (b).

9. The method of claim 8, wherein the DTwPH combination vaccine

has a final pH of 6.5-7.5.

10. The method of any of claims 8 and 9, wherein the concentration of

aluminum hydroxide gel in the DTwPH combination vaccine is adjusted in the range

15-35//gAl/mL.

11. The method of any of claims 8 through 10, wherein the

concentration of diphtheria toxoid is adjusted in the range of 20-50 Lf/mL, the

concentration of tetanus toxoid is adjusted in the range of 4-20 Lf/mL, the

concentration of pertussis is adjusted in the range of 10-24 OU/mL, the concentration

of Hepatitis B surface antigen is adjusted in the range of 10-30 βg/mL, and total

aluminum gel concentration is adjusted in the range of 550-1000 βgAVmL.

12. A combination vaccine storage method in which a combination

vaccine is portioned into and stored in a silicon-coated glass bottle.

13. The method of claim 12, wherein the combination vaccine is a

DTwPH combination vaccine against diphtheria, tetanus, pertussis, and hepatitis type

B.