Donthamsetty Nageswara Rao

Developing subunit immunogens using B and T cell epitopes and their constructs derived from the F1 antigen of Yersinia pestis using novel delivery vehicles

Yersinia pestis is the etiological agent of pneumonic and bubonic plague. As the currently licensed vaccines for plague have their own limitations, there is a need for a rational and more effective form of a subunit vaccine to combat both forms of the disease. Newer methods of antigen delivery coupled with adjuvant offer an alternative approach toward a plague vaccine. In order to develop a new generation vaccine against plague, we chose an immunodominant, outer membrane capsular protein, F1 of Y. pestis. The immunogenicity of the peptide sequences, predicted to possess B (three sequences, B1, B2 and B3) and T (two sequences, T1 and T2) cell determinants, was studied in a murine model with different genetic backgrounds, using alhydrogel and liposomes as delivery vehicles. All the peptide sequences are immunogenic in all mouse strains and showed primary and secondary immune response. B2 peptide was found to be most immunogenic, followed by B1 and B3 peptides. Chimeras made between B and T structures proved highly immunogenic and the antibody levels are comparable with native F1 antigen, thereby proving that T1 and T2 are helper sequences. Interestingly, the liposome mode of immunization was found to be more immunogenic and generated higher affinity antibodies than the alum-based preparation. Immunization using a mixture of all the peptides further proved B2 to be immunodominant. The IgG isotype profile showed predominance of IgG1, IgG2b followed by IgG2a for all the formulations irrespective of mode of antigen delivery. Lymphocyte proliferation of spleen cells primed in vivo with peptides, B-T conjugates and F1 antigen followed by in vitro stimulation with these antigens in soluble (medium) and particulate (liposome) form, showed dose-dependent stimulation of T cells, while B-T constructs showed a higher stimulation index, comparable to F1 antigen. The liposome mode of antigen presentation showed higher lymphoproliferation of spleen cells. Of all the peptides tested, T1 and T2 sequences showed the highest stimulation indices. The pattern of cytokine levels was in the following order: interferon-gamma>interleukin-2>interleukin-4. In vivo protective studies of the B-T conjugates revealed that B1T1 and a mixture of conjugates showed a survival rate of 10 days. Thus, the study highlights the importance of B and T cell epitopes as peptide-based immunogens, being a serious alternative for plague vaccine.

Construction of a synthetic immunogen: use of the natural immunomodulator polytuftsin in malaria vaccines against RESA antigen of Plasmodium falciparum.

Polytuftsin, a 35-40-unit repeat of the naturally occurring tetrapeptide tuftsin (TKPR), was chemically linked to EENVEHDA and DDEHVEEPTVA repeat sequences of ring-infected erythrocyte surface antigen protein (an asexual blood-stage antigen) of Plasmodium falciparum. These synthetic constructs were tested for their humoral and cellular immune responses in five inbred strains of mice with different genetic backgrounds (H-2a, H-2b, H-2d, H-2k and H-2i). Mice immunized with these constructs showed higher antibody titres, secondary immune responses and antigen-induced T-cell proliferation compared with the peptide dimers alone. Sera from mice immunized with both the constructs inhibited merozoite invasion of erythrocytes in vitro by 60-80% at 1:10 antisera dilution. Polytuftsin alone proved to be a very poor immunogen in our studies, since no anti-tuftsin antibodies could be detected in the sera. Therefore, we conclude that the synthetic constructs described here could be useful for the development of subunit malaria vaccines.

Evaluation of synthetic peptides of WbSXP-1 for the diagnosis of human lymphatic filariasis

Parasitic nematodes infect nearly half of the worlds human population, resulting in significant morbidity and mortality. Though filariasis is not fatal, it is the second leading cause of permanent and long-term disability worldwide. Filariasis has a spectrum of disease manifestation and infectivity found among the infected individuals and also goes unnoticed for years. Furthermore, there are ample reports emerging on the genetic variation among the parasites population. Hence, it is necessary to develop diagnostics for early detection of the disease. Synthetic peptides that mimic the immunogenic regions and a conserved region similar to that of recombinant antigen will be more useful in developing diagnostics, vaccines, or therapeutics. WbSXP-1 was earlier proven as a good diagnostic antigen; B-cell epitopic analysis showed 4 potent immunodominant regions spanning the whole antigen. These synthetic peptides (N, N1, N2, and N3) were produced and used as a diagnostic candidate to detect anti-SXP antibody and conversely to detect the infected individuals. The monomeric peptides showed good reactivity against microfilareamic (MF) sera. Among them, the peptides N, N1, and N2 were found to be more reactive. Furthermore, multiple chimeric peptides in linear combinations of 2 peptides were tested for its efficacy to detect anti-SXP antibody in infected MF sera. The peptides N:N1 and N1:N2 were synthesized and tested against human clinical sera. This chimeric peptides constructed based on WbSXP-1 were found to be reactive, specifically with MF sera by ELISA. These peptide-based diagnostic method can serve as a standard better tool without cross-reactivity in lymphatic filariasis elimination program.

Cell‐Mediated Immune Response and Th1/Th2 Cytokine Profile of B‐T Constructs of F1 and V Antigen of Yersinia pestis

Yersinia pestis, a Gram‐negative bacterium, is the etiological agent of pneumonic and bubonic plague and still active in various regions of the world. Because plague is highly infectious and can readily spread by aerosolization, it poses a bioterrorism threat. The effective induction of mucosal as well as systemic immunity is an important attribute of an improved vaccine for plague. An alternative approach described here is the use of protective epitopes derived from immunodominant antigens (F1 and V) of Yersinia pestis. As T‐cell immunity is also a major contributor of protection, microencapsulated B‐T constructs of F1 and V antigen were used to immunize outbred and inbred mice through intranasal route, and lympho‐proliferative response and cytokine profile of both Th1 and Th2 arms were measured in spleen, lamina propria and Peyer’s patches. Three B‐T constructs of F1 antigen and seven of V antigen showed significantly high T‐cell response in terms of inducing systemic as well as mucosal response when compared to constituent peptides. These ten conjugates showed Th1 cytokine profile whereas rest of the conjugates showed mixed Th1/Th2 response. Four conjugates of V antigen showed high level of IL‐10 production. In present study, microencapsulated B‐T constructs after intranasal immunization generated systemic as well as mucosal immune response in all three sites, which offers an alternative approach for plague vaccine.

Acquired immune response to defined Plasmodium vivax antigens in individuals residing in northern India

In this investigation, we evaluated the naturally acquired immune response to Plasmodium vivax stage-specific antigens in individuals of different age groups belonging to malaria endemic areas of northern India. Four synthetic peptides containing both B- and T-cell epitopes from P. vivax circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen-1 were used to determine both humoral and cellular immune responses. Immunity, in terms of antibody response and T-cell proliferation against these stage-specific peptides, has been observed in the study subjects. The results demonstrated age-dependent antibody response in this population. Forty two patients were diagnosed with P. vivax. There was a significant association (P=0.013) between number of antibody responders and recognition of stage-specific epitopes by antibodies. The antibody response to B-epitopes of P. vivax CSP, MSP1, AMA1 and GAM1 was associated with age; adults responded more frequently to these antigens than did younger children. In this population, 66% (201/304) cases showed seropositivity to all peptides and 13% (41/304) showed negative response. Peripheral blood mononuclear cells of more than 75% of individuals proliferated in response to stimulation by all four epitopes. In conclusion, the results demonstrated immunogenicity of the epitopes to P. vivax in population of this endemic zone.

Influence of immunoadjuvants and a promiscous T-cell determinant on the immunogenicity of RESA peptide antigen of P. falciparum.

Synthetic peptide antigens representing the repeat sequences of malarial antigens showed poor immunogenicity and protection in clinical trials. In the present study, RESA, an asexual blood stage antigen, containing (EENVEHDA)2 and (DDEHVEEPTVA)2 sequences were chemically linked to a promiscous T-cell determinant (CS.T3) of the circumsporozoite protein of P. falciparum. The synthetic constructs either alone or coentrapped with immunoadjuvants (nor muramyl dipeptide/lauroyl tetrapeptide) were administered in liposomes to mice of varying genetic background and the immunogenicity of different formulations were compared under identical experimental conditions. The RESA peptide formulations containing the T-cell determinant and the adjuvants generated high titre and affinity antibodies in all the strains, as compared to peptide(s) alone. The booster immunization induced a strong anamnestic response in each group. Though the major IgG isotype is of IgG1 and IgG2b interestingly, formulations containing CS.T3 have a higher proportion of cytophilic IgG2b isotype. There was a significant fall in the levels of IgG2b isotype while IgG1 levels were maintained same in the third bleed (day 60, without booster immunization). The mixed peptide group preparation containing the adjuvant is found to be a better immunogen than that of respective peptides itself. The in vitro merozoite reinvasion inhibition assay showed 76-96% inhibition with the formulations containing RESA peptide(s)-CS.T3 and the adjuvant, while with peptides alone the inhibition was 50-56%. This study highlights the importance of an alternative approach for developing peptide based immunogen against malaria.

Enteromorpha compressa (L.) Greville an edible green alga as a source of antiallergic principle (S)

Enteromorpha compressa a marine green algal species grows extensively in North coastal Andhra Pradesh. Besides its nutritional importance it has also been identified as source of anti-anaphylactic compound(s).E. compressa extracts alleviated the IgE levels raised against ovalbumin and other allergens in mice. Further,Enteromorpha extract also significantly down regulated the serum IgE levels in different murine models irrespective of their genetic background. The results obtained in this study suggest thatE. compressa extract has compound(s), which inhibit IgE immune response and may have potential in curing various types of allergies.

Modulation of the humoral response to repeat and non-repeat sequences of the circumsporozoite protein of Plasmodium vivax using novel adjuvant and delivery systems.

In the use of sub-unit vaccines, it is important to identify the protective epitopes and to generate the optimal immune response by using appropriate immuno-modulatory adjuvants and/or delivery systems. The main aim of the present study was to generate an MHC-non-restricted immune response against one promising vaccine candidate, the circumsporozoite protein (CSP) of Plasmodium vivax. Four synthetic peptides were chosen: three repeat-region sequences (AA, DA and ANG) and a putative T-cell epitope extended from a conserved region (region II) containing a hepatocyte-binding region (HBP). The humoral response against each peptide was studied in outbred mice and three strains of inbred mice (with different genetic backgrounds). Delivery of each peptide in microspheres or inclusion of a bio-active casein-fragment analogue as adjuvant with alum/liposome delivery considerably enhanced the humoral response against the peptide (when compared with the response to the peptide delivered in alum alone). The maximal immune response was observed when the peptide was delivered in microspheres, with no booster doses required; the antibodies raised against peptide delivered with adjuvant or in modulatory delivery vehicles had two-to five-fold lower binding affinities. The predominant IgG isotypes elicited using microspheres or adjuvant with alum/liposome delivery were IgG(2a)/IgG(2b) and/or IgG(1). Importantly, conjugation of HBP to the B-cell repeat peptides increased the titres of peptide-specific antibodies, especially of antibodies against the supposedly cryptic HBP. Delivery of a mix of all four peptides in microspheres elicited an intense immune response in outbred mice, indicating that such a delivery system efficiently presents the peptides to the immune effector cells. That antibodies in the anti-peptide sera bound strongly to air-dried sporozoites of P. vivax was confirmed by immunofluorescence. The present results, based on the use of individual peptides or a conjugate or cocktail of the peptides, highlight the utility of the casein-fragment analogue as an adjuvant, when used with alum/liposome delivery, and also demonstrate the potential of microspheres as a single-shot delivery system for sub-unit peptides.

Crucial epitopes of Wuchereria bancrofti abundant larval transcript recognized in natural infection

Lymphatic filariasis is a tropical disease, with over 40 million people seriously incapacitated due to lymphangitis and elephantiasis. Over 99% of infections are caused by the nematode Wuchereria bancrofti. Expressed sequence tag (EST) analysis of filarial genome identified novel infective larval (L3) stage-specific antigen, abundant larval transcript (ALT-2), which was shown to be highly essential for parasite establishment and survival in the host. The unique structural features and immunological characteristics of ALT-2 indicate the presence of critical motifs that needs to be explored in natural human infection for understanding and management of the disease. In order to dissect the epitopes of ALT protein recognized in humans, eight peptides spanning the entire protein sequence were selected based on in-silico epitope prediction and synthesized. Analysis of the reactivity of W. bancrofti ALT-2 synthetic peptides with clinical sera (n = 40) from endemic areas identified epitopes recognized by putatively immune sera, of which two comprise the highly variable acidic domain (21–60). Interestingly, our study also revealed crucial epitopes recognized by microfilaremic (MF) sera with significantly high IgG4 isotype (p < 0.001), implicated in immunomodulation. The epitope peptides identified were highly specific for MF sera and, thus, have the potential to be exploited as diagnostic markers.

Humoral immune responses and protective efficacy of sequential B- and T-cell epitopes of V antigen of Yersinia pestis by intranasal immunization in microparticles

Capsular F1 and secretory V antigen are the putative vaccine candidates for plague, caused by Yersinia pestis. Contemplating this, we studied the immunogenicity and protective efficacy of collinearly synthesized B- and T-cell epitopes (B-T constructs) of V antigen entrapped in poly (dl-lactide-co-glycolide) microparticles immunized intranasally using single dose immunization schedule in outbred, H-2b and H-2d mice. High antibody levels were observed in terms of IgG, IgA and SIgA peak titers in sera and mucosal washes to different B-T constructs. The constructs ai, bi and fi especially showed high peak antibody titers ranging from 51,200 to 204,000, which were maintained till day 120 post immunization. IgG/IgA Specific activity in sera and washes correlated well with the peak antibody titers. Moreover, all the B-T constructs showed mixed IgG1 and IgG2a/2b response, variable immunoreactivity as well as memory response with V antigen. B-T constructs, viz ai, ak, bi, fi, di and ik showed comparatively high isotype levels. These constructs showed high immunoreactivity, and good recall response with V antigen. Finally, in vivo protective study in BALB/c mice demonstrated the protective efficacy of three B-T constructs (ai, bi and fi) against lethal doses of Yersinia pestis till day 20 post challenge, while construct ‘id’ showed partial protection.