Influence of Different Antigen Administrations on the Production of

Antibodies against Clostridium perfringens Beta Toxin

Yiwen Zhu

Vista Del Lago High School, Folsom, California 95630, U.S.A.

Keywords: Intermuscular, Intraperitoneal, Intradermal, DNA Vaccine, Homogenous Boost.

Abstract: Clostridium perfringens beta toxin (CPB) is a toxin produced by C.perfringens that is responsible for

hypertension and necrotic enteritis in mammals. Currently, DNA vaccines have been implemented to avoid

such toxins. Previous studies have shown that heterologous prime boosting helps with the identification of

CPB by producing more antibodies. However, there was no suggestion that differences in the administration

of antibodies can further influence the production of antibodies. The study focuses on the optimal injection

administration of DNA vaccine and the heterologous protein boost targeting CPB. The study provides

important information to maximize the efficiency of immune response against CPB from the vaccine for

mammals.

1 INTRODUCTION

Th1 and Th2 cells are two types of T helper cells

which leads to an increase in cell-mediated response

against virus, bacteria, and other possible pathogens

(Kidd 2003). Specifically, Th1 cells produce

interferon-gamma, interleukin -2, and tumor necrosis

factor (TNF). Th2 cells produce IL-4, IL-5, IL-10, IL-

13 (Romagnani 1999). These cytokines are

responsible for a strong antibody production and the

activation of eosinophil. These two immune cells are

essential to the adaptive immune system to carry out

its goal in fighting off infections.

Clostridium perfringens beta toxin (CPB) is

responsible for necrotic enteritis, a disease that can be

seen on humans after consuming unclean meat, in

mammals and causes hypertension. It is one of the

four major toxins produced by Clostridium

perfringens. Recent studies have shown that by using

a heterologous prime-boosting strategy in the DNA

vaccine, the amount of anti-rCPB antibody response

was stronger in comparison to a homologous boosting

strategy. Heterologous strategy triggers both Th1 and

Th2 response and the balance in response was

necessary for the success of the vaccine. A possible

link to the success is the increase in both levels of

IgG1 and IgG2b antibodies in comparison to the

IgG2b only reaction in homologous boosting

response. However, there was no evidence suggesting

that the administration of antigens influences the

immune response by resulting in a better

identification and neutralization of the toxin (Solanki,

A. K., Bhatia, B., Kaushik, H., Deshmukh, S. K.,

Dixit, A., & Garg, L. C. 2017). Previous study has

shown that Th1 and Th2 immune responses are not

strictly synchronized because Th2 immune responses

suppress the activities of phagocytosis. Under the

infection of large eukaryotic pathogens, Th1 cell

response can be seen as protective and Th2 cell

response would resolve the inflammation

(Romagnani 1999).

There are seven discovered routes which antigen

administration has in the humoral immune response:

intravenous, intraperitoneal, intermuscular,

intranodal, intrasplenic, intradermal, and

subcutaneous. This study will focus on

intraperitoneal, intermuscular, and intradermal

routes. Previous study suggests that different antigen

administrations have no significant influence on

antibody production. I hypothesize that the

introduction of vaccine into the host of clostridium

perfringens beta toxin will result in most

improvement in the production of antibodies

measured by ELISA and a better identification and

neutralization of the toxin after immunization

through the intradermal, following intramuscular, and

lastly intraperitoneal.

716

Zhu, Y.

Inﬂuence of Different Antigen Administrations on the Production of Antibodies against Clostridium perfringens Beta Toxin.

DOI: 10.5220/0011263800003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 716-722

ISBN: 978-989-758-595-1

2 MATERIALS

2.1 Cells Used in the Study

E. coli DH5α and BL21 pLysS cells were used in the

study along with DNA information and modifying

enzymes.

2.2 Genetic Information of the CPB

Genetic information of the beta toxin in this study

was used to optimize reactions. Genetic information

will also be later used to introduce the booster shot in

all mices. In order to fit the large requirements in this

study. This piece of genetic information will be

replicated through the process of PCR.

2.3 ELISA (Enzyme-Linked

Immunosorbent Assay)

The antigen-specific antibodies of each group will be

read by ELISA. In the study the antibody will be

specific to Anti-rCPB antibodies.

2.4 Mice Immunization

In this study, each experimental group will contain 3

mice. Each mice will be immunized with 100ug of

plasmid DNA following a booster shot of rCPB on

day 15 after immunization. There will be a total of 29

groups of mice in this study to examine specific

differences. They will be the 27 possible results

presented by the different combinations of antigen

administrations along with positive control group and

negative control group. The positive control group

will receive the inactivated bacteria. In addition, the

negative control group will receive saline at all

routes.

2.5 Data Analysis

Significance of all levels of antibody production

measured by ELISA will be analyzed on TI-84 Plus

CE. (p<0.05)

3 RESULTS

Possible results on the production of antibodies for

different antigen administrations. All 27 possible

results can be seen at Table 1 Different Antibody

Production Measured by ELISA.

3.1 Possible Result #1

DNA vaccine administered through the

intramuscular route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will not

produce any antibodies against CPB.

3.2 Possible Result #2

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will not

produce any antibodies against CPB.

3.3 Possible Result #3

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will not

produce any antibodies against CPB.

3.4 Possible Result #4

DNA vaccine administered through the

intramuscular route, receiving booster shot

through the intradermal route. The production of

antibodies in these specific combinations will not

produce any antibodies against CPB.

3.5 Possible Result #5

DNA vaccine administered through the

intramuscular route, receiving the booster shot

through the intraperitoneal route. The production

of antibodies in these specific combinations will not

produce any antibodies against CPB.

3.6 Possible Result #6

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the intramuscular route. The production

of antibodies in these specific combinations will not

produce any antibodies against CPB.

3.7 Possible Result #7

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the intradermal route. The production of

antibodies in these specific combinations will not

Inﬂuence of Different Antigen Administrations on the Production of Antibodies against Clostridium perfringens Beta Toxin

717

produce any antibodies against CPB.

3.8 Possible Result #8

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the intramuscular route. The production

of antibodies in these specific combinations will not

produce any antibodies against CPB.

3.9 Possible Result #9

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the intraperitoneal route. The production

of antibodies in these specific combinations will not

produce any antibodies against CPB.

3.10 Possible Result #10

DNA vaccine administered through the

intramuscular route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.11 Possible Result #11

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.12 Possible Result #12

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.13 Possible Result #13

DNA vaccine administered through the

intramuscular route, receiving booster shot

through the intradermal route. The production of

antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.14 Possible Result #14

DNA vaccine administered through the

intramuscular route, receiving the booster shot

through the intraperitoneal route. The production

of antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.15 Possible Result #15

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the intramuscular route. The production

of antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.16 Possible Result #16

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the intradermal route. The production of

antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.17 Possible Result #17

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the intramuscular route. The production

of antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.18 Possible Result #18

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the intraperitoneal route. The production

of antibodies in these specific combinations will

produce high enough antibodies against CPB to

efficiently eliminate the bacteria.

3.19 Possible Result #19

DNA vaccine administered through the

intramuscular route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

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3.20 Possible Result #20

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

3.21 Possible Result #21

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the same route. The production of

antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

3.22 Possible Result #22

DNA vaccine administered through the

intramuscular route, receiving booster shot

through the intradermal route. The production of

antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

3.23 Possible Result #23

DNA vaccine administered through the

intramuscular route, receiving the booster shot

through the intraperitoneal route. The production

of antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

3.24 Possible Result #24

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the intramuscular route. The production

of antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

3.25 Possible Result #25

DNA vaccine administered through the

intraperitoneal route, receiving the booster shot

through the intradermal route. The production of

antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

3.26 Possible Result #26

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the intramuscular route. The production

of antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

3.27 Possible Result #27

DNA vaccine administered through the

intradermal route, receiving the booster shot

through the intraperitoneal route. The production

of antibodies in these specific combinations will

produce a low amount of antibodies against CPB that

doesn’t support an efficient response to eliminate the

bacteria.

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Table 1 Different Antibody Production Measured by ELISA

4 DISCUSSION

Previous studies have shown that there would be an

increase in immune response when an extra booster

shot is injected in addition to the shot of DNA vaccine

targeting CPB. This study applied the ELISA method

to examine how the production of antibodies can be

influenced by different methods of administration.

The hypothesis made for this experiment is based on

a logical assumption. Intradermal injection is not

common for vaccination because it requires a difficult

technique which slows down the efficiency to

vaccinate the population. However, areas of

intradermal injection generally contain large amounts

of dendritic cells, which are immune cells that present

antigens to the adaptive immune system, where

antibodies are produced. However, with livestock

vaccination, it would be more ideal than

intramuscular injection since some mammals have a

thick skin layer. Therefore, I hypothesize that

intradermal would be the most effective out of the

three routes. Intermuscular, or the injection of

vaccine in the muscle, is predicted to have less

antibody production when compared to intradermal

injection because muscles have more blood supplies

which indicates that as cytokines are released, it will

be easier for neutrophils to reach the site of injection

and process the materials injected into the muscles

and absorb the information. The least effective

method of three would be intraperitoneal for various

reasons. This injection method is largely used in mice

rather than humans or larger cattles since larger

mammals would require a larger needle to be able to

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reach the abdomen. In addition, this injection route is

usually for a large concentration of fluid which is not

a typical feature of vaccines. Booster injections may

also play a role in the production of antibodies. Since

the immune system would already have memories of

CPB after the injection of the vaccine, it would be the

same logic as the initial exposure to antigen, placing

the injection site at a location with easier absorption

and presentation of antigens would result in the most

production of antibodies.

The possible results that are provided by Table 1.

It presents three different possible results in the

production of antibodies for a specific injection

combination. When there are no antibodies produced,

the possible explanation is that the specific injection

site doesn’t trigger the immune system at all.

Previous studies proved that the DNA Vaccine would

trigger an immune response and produce specific

antibodies against the toxin (Solanki, A. K., Bhatia,

B., Kaushik, H., Deshmukh, S. K., Dixit, A., & Garg,

L. C. 2017). However, if the method did not produce

any antibodies for all mice in a group with the same

route for both the injection of booster protein and

vaccine, it signifies that the route somehow does not

trigger an immune response inside the mice, meaning

that the immune cells involved in the innate immune

response does not recognize any pathogen and

thereby not presenting any antigen for the production

of antibodies. I believe that such a situation is not

likely. However, there is no evidence to prove the

nonexistence of such possibility nor evidence of the

principle behind the observation.

When looking at the result, the results of mice

groups of the two routes that are identical to each of

the pathways should be compared. For instance, the

results of possible result #14 presents two different

routes: intramuscular and intraperitoneal. Hence the

production of antibodies of result #1 and 3 should be

compared with result #14. If both of them function

properly, this data signifies that the route of injection

is able to effectively trigger an immune response as

protein or vaccine is injected at the site.

On the other hand, it would be obvious that there

are other factors contributing to the lack of antibodies

produced. Similar to the first observation, such a

result is unlikely to occur. There is also the possibility

that no antibody binded to the plate in ELISA.

However, since there are multiple serums of mice

being tested, it is quite unlikely that no antibody

attached to any of the receptors in the plates.

Other possible observations of the presence of

produced antibodies against CPB are much more

likely to occur in reality. If a general trend is

discovered in the difference between each

combination of antigen administrations, specifically

the difference between a high production and a low

production, it is possible that the observation is a

result of the efficient connection between the antigen

receiving B cells and the activated B cells that are

responsible for the production of antibodies.

However, each mammal may have different features

of the immune system. It is not possible to test an

individual mammal multiple times on the specific

vaccines since the immune system would already

have memory of the antigen. Therefore, antibody

production using a separate vaccine on the same

mammal can be observed to discover if the low

production of anti-CPB is a result of ineffective

delivery of antigen information or of injection sites.

5 CONCLUSION

This study explores the influences of different

injection administration on the production of

antibodies against Clostridium perfringens beta toxin.

It is predicted that intradermal injection will produce

the most amount of antibodies, followed by

intramuscular injection having the second most

production and intraperitoneal being the least

effective. The possible results predict the potential

relationship between the identification of antigen

after initial injection and the delivery of information

in the immune system. Since the hypothesis made in

this study can only be directly supported or rejected

with numbers, Table 1 suggests that results #1, 3, 5,

6, 11, 13, 16, 17, 18, 19, 21, 23, and 24 all partially

support the hypothesis and #2, 4, 7, 8. 9. 10. 12. 14,

15, 20, 22, 25, 26, 27 all partially rejected the

hypothesis. In addition, since the use of DNA vaccine

has been prevalent on livestock to prevent economic

losses, the result of the study will be able to advance

the efficiency of vaccination. However, since there

are more than three injection administrations and

different animals support different injection methods,

further comparison between all the routes is

necessary before concluding the most efficient

method for the vaccination of Clostridium

perfringens beta toxin on different mammal species.

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