Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.     

Protective antigens of rodent and human bloodstage malaria

Bloodstage malaria parasites are antigenically complex, but individual antigens can be identified and analysed using monoclonal antibodies. Two monoclonal antibodies that recognize a 235 000 molecular mass Plasmodium yoelii rhoptry protein provide some protection when injected into mice against a challenge infection. The purified rhoptry protein also provides protective immunity against P. yoelii YM when used to vaccinate mice and fulminating infections are converted into self-limiting, reticulocyte-restricted infections. Another monoclonal antibody immunoprecipitates a 230 000 molecular mass protein and a series of proteolytic processing fragments. At least one of these processing fragments, probably a 90 000 molecular mass species, is located on the merozoite surface. Mice immunized with the purified protein were protected against challenge infection with P. yoelii YM. This antigen may provide protection by inducing a cell-mediated immune response. A monoclonal antibody raised against P. falciparum schizonts reacts with a 195 000 molecular mass protein which is synthesized in schizonts and subsequently cleaved. Fragments of the 195 000 molecular mass protein are expressed as major antigens on the merozoite surface. The 195 000 molecular mass P. falciparum protein and the 230 000 molecular mass P. yoelii protein belong to a class of malaria parasite antigens which probably is important in the induction of a protective immune response in the host.     

Effect of adjuvants on immune response and protective immunity elicited by recombinant Hsp60 (GroEL) of Salmonella typhi against S. typhi infection

Heat shock proteins (Hsps) have been reported to be dominant antigens for the host immune response to various pathogens and thus, have great potential for use in vaccination. In the present study, we evaluated the immunogenicity and protective efficacy of GroEL of Salmonella enterica serovar Typhi against lethal infection by S. typhi Ty2 in mice with or without adjuvants. Anti GroEL–IgG titers were significantly higher in mice immunized with either GroEL-alone or in combination with alum/Complete Freund’s adjuvant (CFA) as compared to the control. Analysis of antibody isotypes suggested predominance of Th2 type immune response in GroEL + alum immunized animals as revealed by higher IgG1/IgG2a ratio. Whereas, immunization of animals with GroEL + CFA or GroEL-alone shifted the immune response toward Th1 phenotype. Mice immunized with GroEL with or without adjuvants, showed a significant increase in lymphocyte proliferation and cytokine levels. The animals immunized with GroEL + CFA or GroEL-alone showed higher IFN-γ and IL-2 levels than alum group, indicating Th1 response whereas IL-4 levels (Th2 response) were found to be highest in alum group as compared to other two immunized groups. Immunization of mice with GroEL-alone, GroEL + alum, and GroEL + CFA provided 70, 50 and 80% protection, respectively, against lethal challenge by S. typhi in mice. The differences in the percentage protection among various groups were attributed to the differences in the immune responses generated by respective immunizations. The present study shows that GroEL forms an ideal candidate molecule to develop a recombinant protein based vaccine against human typhoid.     

Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72

The use of anti-idiotypic antibodies as immunogens represents one potential approach to active specific immunotherapy of cancer. Two panels of syngeneic monoclonal anti-idiotypic antibodies were generated. One panel was directed against mAb CC49 and the other to mAb COL-1. mAb CC49 recognizes the pancarcinoma antigen (Ag), tumor-associated glycoprotein-72 (TAG-72), and mAb COL-1 recognizes carcinoembryonic antigen (CEA). Seven anti-idiotypic (AI) antibodies (Ab2) designated AI49-1–7 were generated that recognize the variable region of mAb CC49. These mAb were shown to inhibit the interaction of mAb CC49 (Ab1) with TAG-72 (Ag). Five anti-idiotypic antibodies designated CAI-1–5 were also generated to the anti-CEA mAb, COL-1 (Ab1). These Ab2 were shown to inhibit the interaction between COL-1 (Ab1) and CEA (Ag). Immunization of mice, rats, and rabbits with Ab2 directed against CC49 or COL-1 could not elicit specific Ab3 humoral immune responses, i.e., antibody selectively reactive with their respective target antigens. However, immunization of mice with the CC49 anti-idiotypic antibody (Ab2), designated AI49-3, could induce a delayed-type hypersensitivity response (DTH) specific for tumor cells that express TAG-72. Similarly, immunization of mice with an anti-idiotypic antibody directed against COL-1, designated CAI-1, could induce specific DTH cell-mediated immune responses to murine tumor cells that express human CEA on their surface. These results thus demonstrate that while some anti-idiotype mAb may not be potent immunogens in eliciting Ab3 humoral responses, they are capable of eliciting specific cellular immune responses against human carcinoma-associated antigens. This type of mAb may ultimately be useful in active immunotherapy protocols for human carcinoma.Some of the studies described in this paper were in partial fulfillment of requirements for the completion of Dr. Irvine's dissertation at the George Washington University     

IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice

Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti- N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis . IgG mAb lacked this protective effect. IgM mAb showed a dose–response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.     

结核分枝杆菌蛋白亚单位疫苗与疫苗诱导的T细胞免疫记忆研究进展

牛分枝杆菌减毒活疫苗--卡介苗(bacillus Calmette-Guérin,BCG)对预防严重的儿童结核病有效,但其免疫保护效率随儿童年龄增长而降低。BCG不能提供终身免疫保护可能与其诱导的记忆性T细胞主要是寿命较短的效应记忆性T细胞有关。新型结核分枝杆菌蛋白亚单位疫苗将有效的抗原有机组合起来,在适宜的疫苗佐剂辅助下诱导Th1型免疫应答。动物实验表明,增加抗原谱可有效提高亚单位疫苗的保护效率。更重要的是,亚单位疫苗在体内持续时间较短,可诱导寿命较长的中央记忆性T细胞,提供比BCG更持久的免疫保护力。记忆性T细胞的分化受抗原特性与剂量、细胞因子、转录因子及雷帕霉素等的调控。对亚单位疫苗及其诱导的免疫记忆进行研究将对新型结核分枝杆菌疫苗的设计与评价产生积极影响。     

Type I IFN as a natural adjuvant for a protective immune response: lessons from the influenza vaccine model

The identification of natural adjuvants capable of selectively promoting an efficient immune response against infectious agents would represent an important advance in immunology, with direct implications for vaccine development, whose progress is generally hampered by the difficulties in defining powerful synthetic adjuvants suitable for clinical use. Here, we demonstrate that endogenous type I IFN is necessary for the Th1 type of immune response induced by typical adjuvants in mice and that IFN itself is an unexpectedly powerful adjuvant when administered with the human influenza vaccine, for inducing IgG2a and IgA production and conferring protection from virus challenge. The finding that these cytokines, currently used in patients, are necessary for full expression of adjuvant activity and are sufficient for the generation of a protective immune response opens new perspectives in understanding the basis of immunity and in vaccine development.     

Immunogenicity of Leishmania donovani iron superoxide dismutase B1 and peroxidoxin 4 in BALB/c mice: the contribution of Toll-like receptor agonists as adjuvant

In this study, we assessed the immune response of two Leishmania donovani recombinant proteins: iron superoxide dismutase B1 (SODB1) and peroxidoxin 4 (Pxn4) in BALB/c mice. Assessment of the immunogenicity of these proteins alone or combined with Toll-like receptor 9 (TLR-9) agonist (CpG ODN) or TLR-4 agonist (GLA-SE) showed that they elicit specific antibody as well as cytokine production in response to the respective antigen in vitro. The use of adjuvants augmented immunogenicity of these antigens and more importantly, skewed the immune response to a Th1-type. These results indicate that recombinant SODB1 and Pxn4 proteins are potential vaccine candidates when administered with appropriate adjuvants.     

Trichinella spiralis: Immune response and protective immunity elicited by recombinant paramyosin formulated with different adjuvants

Our previous studies showed that immunization with recombinant paramyosin from Trichinella spiralis (rTs-Pmy) formulated with Freund’s adjuvant significantly reduced larval burden in mice after T. spiralis larval challenge. Since Freund’s adjuvant is toxic and not a suitable adjuvant for clinical vaccine trials, we evaluated the ability of the adjuvants Montanide ISA206 and ISA720 to stimulate immune responses during rTs-Pmy immunization and to enhance protective immunity. The results revealed that immunization of BALB/c mice with rTs-Pmy formulated with either ISA206 or ISA720 triggered Th1 and Th2 immune responses similar to those produced by the conventional Freund’s adjuvant formulation and also provided a similar level of protection against T. spiralis larval challenge. This indicates that the recombinant Ts-Pmy formulated with Montanide ISA206 or ISA720 may be an effective and safety vaccine strategy for trichinellosis.     

A malaria vaccine based on the polymorphic block 2 region of MSP-1 that elicits a broad serotype-spanning immune response

Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen.     

Active and passive immunization of mice against larval Dirofilaria immitis

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.     

Immunostimulatory DNA pre-priming: a novel approach for prolonged Th1-biased immunity

Immunostimulatory DNA sequences (ISS) have been shown to promote CTL and Th1 immune responses to coinjected antigens. This phenomenon can be attributed to the capacity of ISS to induce the secretion of type-1 cytokines and to up regulate costimulatory ligands on antigen-presenting cells. We hypothesized that ISS administration prior to antigen administration (prepriming), via the mechanisms stated above, would Th1 bias immune responses to subsequently injected antigens for an extended period of time. The data presented show that ISS provide in vivo adjuvant activity for up to 2 weeks after intradermal or intranasal delivery. Furthermore, the results demonstrate that ISS prepriming can induce immune responses that are significantly stronger than with ISS/antigen covaccination. ISS prepriming offers an alternative approach to the traditional use of adjuvants (i.e., antigen/adjuvant coinjection) and expands the potential clinical applications for ISS.     

Functional Activity of Antibodies Directed towards Flagellin Proteins of Non-Typhoidal Salmonella

Non-typhoidal Salmonella (NTS) serovars Typhimurium and Enteritidis are major causes of invasive bacterial infections in children under 5 years old in sub-Saharan Africa, with case fatality rates of ~20%. There are no licensed NTS vaccines for humans. Vaccines that induce antibodies against a Salmonella Typhi surface antigen, Vi polysaccharide, significantly protect humans against typhoid fever, establishing that immune responses to Salmonella surface antigens can be protective. Flagella proteins, abundant surface antigens in Salmonella serovars that cause human disease, are also powerful immunogens, but the functional capacity of elicited anti-flagellar antibodies and their role in facilitating bacterial clearance has been unclear. We examined the ability of anti-flagellar antibodies to mediate microbial killing by immune system components in-vitro and assessed their role in protecting mice against invasive Salmonella infection. Polyclonal (hyperimmune sera) and monoclonal antibodies raised against phase 1 flagellin proteins of S. Enteritidis and S. Typhimurium facilitated bacterial uptake and killing of the homologous serovar pathogen by phagocytes. Polyclonal anti-flagellar antibodies accompanied by complement also achieved direct bacterial killing. Serum bactericidal activity was restricted to Salmonella serovars expressing the same flagellin used as immunogen. Notably, individual anti-flagellin monoclonal antibodies with complement were not bactericidal, but this biological activity was restored when different monoclonal anti-flagellin antibodies were combined. Passive transfer immunization with a monoclonal IgG antibody specific for phase 1 flagellin from S. Typhimurium protected mice against lethal challenge with a representative African invasive S. Typhimurium strain. These findings have relevance for the use of flagellin proteins in NTS vaccines, and confirm the role of anti-flagellin antibodies as mediators of protective immunity.     

Combined immunization with adjuvant molecules poly(I:C) and anti-CD40 plus a tumor antigen has potent prophylactic and therapeutic antitumor effects

The low immunogenicity of malignant cells is one of the causes responsible for the lack of antitumor immune responses. Thus, development of new therapeutic strategies aimed at enhancing presentation of tumor antigens to T cells is a main goal of cancer immunotherapy. With this aim, we studied the efficacy of administering adjuvants poly(I:C) and agonistic anti-CD40 antibody plus a tumor antigen. Joint intravenous immunization with these adjuvants and a model tumor antigen (ovalbumin) was able to synergistically induce potent and long lasting antitumor T-cell responses. These responses protected against challenge with E.G7–OVA tumor cells in prophylactic short- and long-term vaccination. In a therapeutic setting, repeated intratumor administration of adjuvants plus antigen was able to reject established tumors in all treated animals, leading in some cases to the rejection of both locally treated and untreated tumors. Antitumor immune responses induced by these protocols were mediated not only by T-cells but also by NK cells. In conclusion, combined administration of adjuvants poly(I:C) and anti-CD40 plus a tumor antigen is an efficient strategy for prophylactic and therapeutic antitumor vaccination.     

Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL/6 mice

Background

The current knowledge of immunological responses to schistosomiasis, a major tropical helminthic disease, is insufficient, and a better understanding of these responses would support vaccine development or therapies to control granuloma-associated immunopathology. CD4⁺ T cells play critical roles in both host immune responses against parasitic infection and immunopathology in schistosomiasis. The induction of T helper (Th)1, Th2 and T regulatory (Treg) cells and their roles in schistosome infections are well-illustrated. However, little in vivo data are available on the dynamics of Th17 cells, another important CD4⁺ T cell subset, after Schistosoma japonicum infection or whether these cells and their defining IL-17 cytokine mediate host protective responses early in infection.

Methodology

Levels of Th17 and the other three CD4⁺ T cell subpopulations and the cytokines related to induction or repression of Th17 cell generation in different stages of S. japonicum infection were observed. Contrary to reported in vitro studies, our results showed that the Th17 cells were induced along with the Th1, Th2, Treg cells and the IFN-γ and IL-4 cytokines in S. japonicum infected mice. The results also suggested that S. japonicum egg antigens but not adult worm antigens preferentially induced Th17 cell generation. Furthermore, decreasing IL-17 with a neutralizing anti-IL-17 monoclonal antibody (mAb) increased schistosome-specific antibody levels and partial protection against S. japonicum infection in mice.

Conclusions

Our study is the first to report the dynamics of Th17 cells during S. japonicum infection and indicate that Th17 cell differentiation results from the integrated impact of inducing and suppressive factors promoted by the parasite. Importantly, our findings suggest that lower IL-17 levels may result in favorable host protective responses. This study significantly contributes to the understanding of immunity to schistosomiasis and may aid in developing interventions to protect hosts from infection or restrain immunopathology.     

Uniformity of protective antigens among isolates of the cattle tick, Boophilus microplus

Abstract. Gut membrane antigens were extracted from ten isolates of the cattle tick Boophilus microplus; the antigen extracts were probed with bovine antisera and three murine monoclonal antibodies (mAbs) in Western blots and dot-ELISA. The antisera had been obtained from cattle which were vaccinated with larval and gut extracts of B.microplus , and which were subsequently protected (84% and 94% respectively) against challenge with B.microplus. One of the mAbs (QU13) has been demonstrated to precipitate protective antigens from the midgut of B.microplus. Gut antigens from all ten isolates displayed similar reactivity profiles against bovine antisera and also against mAbs in Western blots. The end-point titres of antigens in dot-ELISA showed four-fold variation between isolates against bovine antisera, and also against mAb QUI 3. Larval membrane antigen extracted from N-strain B.microplus reacted with QU13 in dot-ELISA, indicating that protective antigens are common to both larval and adult stages of B.microplus. It was concluded that protective antigens recognized by QUI3 and antigens recognized by sera from protected cattle were conserved between the ten isolates examined, and between life-cycle stages.     

Adjuvant properties of bacterial product cantastim

Aluminum compounds have been used as adjuvants in practical vaccination for more than 60 years to induce an early, an efficient and a long lasting protective immunity. Nowadays they are the most widely used adjuvants in both veterinary and human vaccines. Unfortunately these adjuvants do not only cause undesirable side effects, but often induce T-helper type 2 (Th2)-biased responses. In this study we investigated the ability of the bacterial product CANTASTIM (CS) to augment the immune responses to a model antigen, tetanus toxoid (TT). Immunization of mice with TT+CS elicited higher anti-TT IgG antibody levels as compared to mice that received TT alone. Moreover, treatment with TT+CS resulted in a lower IgG1/IgG2a ratio and a stronger in vitro IFN-gamma synthesis by splenocytes and T cells cocultured with macrophages. These data suggest that CS can be used to enhance the magnitude of the immune response and to skew it towards the Th1 type.     

Alternative routes of mucosal immunization in large animals

Mucosal immunization regimes that employ the oral route of delivery are often compromised by antigen degradation in the stomach. Moreover, tolerance or immunological unresponsiveness to orally delivered vaccine antigens is also a major problem associated with this route of immunization. Immunization by alternative routes including intrarectal (i.r.) and intranasal (i.n.) is becoming increasingly recognized in large animals for generating protective antibody responses at mucosal surfaces. These approaches are particularly useful in ruminant species which have four stomachs that can potentially interfere with antigen presentation to mucosal inductive sites of the gut. Modifications to enhance existing mucosal immunization regimes have also been explored through the use of alternative antigen delivery systems and mucosal adjuvants. The combination of alternative immunization routes and the use of appropriate antigen delivery systems appear to be a rational approach for providing protective immunity at mucosal surfaces. There has been a considerable amount of research conducted on evaluating the efficacy of emerging antigen delivery systems and novel adjuvants for improved immunity to mucosal immunization but very little of this work has been specific to the mucosal compartment of large animals. The aim of this review is therefore to assess the feasibility and practicality of using large animals (particularly sheep, cattle and pigs) for inducing and detecting specific immune responses to alternative mucosal routes of immunization.     

Local secretion of anti-CTLA-4 enhances the therapeutic efficacy of a cancer immunotherapy with reduced evidence of systemic autoimmunity

Promising anti-tumor responses have been observed in the clinic using monoclonal antibodies (mAbs) that block immune checkpoints. One concern with these therapeutic agents remains the potential induction of immune breakthrough events (IBEs) resulting from the disruption of T cell homeostasis or the breaking of tolerance to self antigens. As an approach to maintaining anti-tumor responses but decreasing the likelihood of these events, the local expression of a mAb in combination with a GM-CSF-secreting cancer immunotherapy was evaluated. Using anti-cytotoxic T lymphocyte antigen (CTLA)-4 as a model antibody to test this hypothesis, tumor cell lines were generated that expressed the full-length mAb in addition to GM-CSF. Evaluation of these cell lines in two therapeutic tumor models revealed that local, cell-mediated delivery of anti-CTLA-4 from a GM-CSF-secreting tumor cell immunotherapy activated potent anti-tumor responses and prolonged overall survival at significantly lower serum mAb levels in the host. Furthermore, lowering the systemic exposure of the host to the immune modulatory mAb correlated with reduced evidence of systemic autoimmunity. This approach has broad utility for the delivery of mAbs or proteins locally from cellular immunotherapies to minimize IBEs while retaining the potent therapeutic effects of such combination treatments.     

Immune response variations to Salmonella enterica serovar Typhi recombinant porin proteins in mice

ObjectivesTyphoid fever is caused by Salmonella enterica serovar Typhi. OmpC, OmpF and OmpA, the three major outer membrane proteins (OMPs), could serve as vaccine candidates.MethodsThe porins antigenicity was predicted in silico. The OMP genes were amplified, cloned and expressed. Sero-reactivities of the recombinant proteins purified by denaturing method were assayed by ELISA. BALB/c mice were immunized with the recombinant porins followed by bacterial challenge.ResultsBacterial challenge of the animal model brought about antibody triggering efficacy of the antigen in OmpF > OmpC > OmpA order. Experimental findings validated the in silico results. None of the antigens had synergic or antagonistic effects on each other from immune system induction points of view. Despite their high immunogenicity, none of the antigens was protective. However, administration of two or three antigens simultaneously resulted in retardation of lethal effect. Porins, in addition to their specific functions, share common functions. Hence, they can compensate for each other's functions.ConclusionsThe produced antibodies could not eliminate the pathogenicity by blockade of one or some of the antigens. Porin antigens are not suitable vaccine candidates alone or in denatured forms. Native forms of the antigens maybe studied for protective immunogenicity.