Synthetic nanoparticle vaccines produced by layer-by-layer assembly of artificial biofilms induce potent protective T-cell and antibody responses in vivo

Nanoparticle vaccines induce potent immune responses in the absence of conventional adjuvant due to the recognition by immune cells of the particle structures, which mimic natural pathogens such as viruses and bacteria. Nanoparticle vaccines were fabricated by constructing artificial biofilms using layer-by-layer (LbL) deposition of oppositely charged polypeptides and target designed peptides on CaCO₃ cores. LbL nanoparticles were efficiently internalized by dendritic cells in vitro by a mechanism that was at least partially phagocytic, and induced DC maturation without triggering secretion of inflammatory cytokines. LbL nanoparticle delivery of designed peptides to DC resulted in potent cross-presentation to CD8+ T-cells and more efficient presentation to CD4+ T-cells compared to presentation of soluble peptide. A single immunization of mice with LbL nanoparticles containing designed peptide induced vigorous T-cell responses characterized by a balanced effector (IFNγ) and Th2 (IL-4) ELISPOT profile and in vivo CTL activity. Mice immunized with LbL nanoparticles bearing ovalbumin-derived designed peptides were protected from challenge with Listeria monocytogenes ectopically expressing ovalbumin, confirming the relevance of the CTL/effector T-cell responses. LbL nanoparticles also elicited antibody responses to the target epitope but not to the matrix components of the nanoparticle, avoiding the vector or carrier affect that hampers utility of other vaccine platforms. The potency and efficacy of LbL nanoparticles administered in aqueous suspension without adjuvant or other formulation additive, and the absence of immune responses to the matrix components, suggest that this strategy may be useful in producing novel vaccines against multiple diseases.     

Differential antibody responses to Plasmodium falciparum-derived B-cell epitopes induced by diepitope multiple antigen peptides (MAP) containing different T-cell epitopes

Epitopes of universal character are needed when designing subunit vaccines against infectious diseases such as malaria. We have compared the immunogenicity of B-cell epitopes from the Plasmodium falciparum antigen repeats DPNANPNV (PfCS protein) and VTEEI (Pf332) when assembled with four different universal T-cell epitopes in diepitope multiple antigen peptides (MAP). T-epitopes employed were from P. falciparum antigens (CS.T3, [T^*]₄ and EBP3) or from the Clostridium tetani toxin (P2). In association with either of the T-epitopes, the genetic unresponsiveness to the B-epitopes was successfully bypassed. Our results show that the immunogenicity of a T-epitope alone does not necessarily predict the ability of the T-epitope to provide T-cell help when combined with other epitopes in an immunogen. Further, the nature of the immune responses in terms of total IgG antibodies and their subclass distribution, T-cell proliferation and IFN-γ production, varied with the T-epitope and mouse strain, which may indicate the need for inclusion of a combination of different universal T-epitopes in a future malaria subunit vaccine.     

Immunoinformatics approach for multi-epitope vaccine design against P. falciparum malaria

Plasmodium falciparum (P. falciparum) is a leading causative agent of malaria, an infectious disease that can be fatal. Unfortunately, control measures are becoming less effective over time. A vaccine is needed to effectively control malaria and lead towards the total elimination of the disease. There have been multiple attempts to develop a vaccine, but to date, none have been certified as appropriate for wide-scale use. In this study, an immunoinformatics method is presented to design a multi-epitope vaccine construct predicted to be effective against P. falciparum malaria. This was done through the prediction of 12 CD4+ T-cell, 10 CD8+ T-cell epitopes and, 1 B-cell epitope which were assessed for predicted high antigenicity, immunogenicity, and non-allergenicity through in silico methods. The Human Leukocyte Antigen (HLA) population coverage showed that the alleles associated with the epitopes accounted for 78.48% of the global population. The CD4+ and CD8+ T-cell epitopes were docked to HLA-DRB1*07:01 and HLA-A*32:01 successfully. Therefore, the epitopes were deemed to be suitable as components of a multi-epitope vaccine construct. Adjuvant RS09 was added to the construct to generate a stronger immune response, as confirmed by an immune system simulation. Finally, the structural stability of the predicted multi-epitope vaccine was assessed using molecular dynamics simulations. The results show a promising vaccine design that should be further synthesised and assessed for its efficacy in an experimental laboratory setting.     

CD8 T cell immunity to Plasmodium permits generation of protective antibodies after repeated sporozoite challenge

Individuals living in malaria endemic areas are subject to repeated infections yet fail to develop sterilizing immunity, however, immunization of mice with attenuated sporozoites or subunit vaccines has shown the ability to protect mice against a sporozoite challenge. We recently reported that mice primed with dendritic cells coated with the dominant circumsporozoite CD8 T cell epitope from Plasmodium berghei followed by a boost with recombinant Listeria monocytogenes expressing the same epitope exhibited sterile immunity against a sporozoite challenge for more than one year. In this report we show those mice do not contain protective antibodies and that depletion of CD4 T cells in the immunized mice did not affect sterile immunity. In contrast, CD8 T cell depletion eliminated protection. Thus, protective immunity generated by this immunization approach is entirely memory CD8 T cell-dependent. We also show here that mice initially protected by circumsporozoite-specific memory CD8 T cells develop sterilizing sporozoite-specific antibodies after repeated asymptomatic challenges with physiologic numbers of viable sporozoites. Therefore, initial protection by a CD8 T cell-targeted liver stage subunit vaccine allows the generation of enhanced sterilizing immune responses from repeated exposure to Plasmodium parasites.     

Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice

A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A10201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A10201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A10201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.     

A potent malaria vaccine based on adenovirus with dual modifications at Hexon and pVII

Adenovirus (Ad) is thought to be one of the most promising platforms for a malaria vaccine targeted against its liver stages, because of its ability to induce a strong T-cell response against a transgene. However, a further improvement of this platform is needed in order to elicit another arm of the immunity, i.e. humoral response, against malaria.In order to augment immunogenicity and protective efficacy of Ad-based malaria vaccine, we inserted B-cell, as well as CD4+ T-cell, epitopes of Plasmodium falciparum circumsporozoite protein (PfCSP) into the capsid protein, Hexon, and the core protein, VII (pVII), of Ad, respectively, in addition to the PfCSP transgene. Insertion of PfCSP-derived B cell epitope to Hexon significantly enhanced the epitope-specific antibody response compared to AdPfCSP, an Ad vaccine expressing only PfCSP transgene. PfCSP-derived CD4+ T-cell epitope insertion into pVII augmented not only PfCSP-specific CD4+ T-cell response but also anti-PfCSP antibody response. Finally, mice immunized with AdPfCSP having both Hexon and pVII modifications were more protected than AdPfCSP or Hexon-modified AdPfCSP against challenge with transgenic rodent malaria parasites expressing the PfCSP.Overall, this study has demonstrated that Hexon and pVII-modified AdPfCSP vaccine is a promising malaria vaccine which induces strong PfCSP-specific humoral, CD4+ T-cell, and CD8+ T-cell responses and protects against infection with transgenic malaria parasites expressing the PfCSP.     

Identification of B- and T-cell epitopes from glycoprotein B of herpes simplex virus 2 and evaluation of their immunogenicity and protection efficacy

Herpes simplex virus (HSV) infection is a major health concern worldwide. Evidence obtained from animals and humans indicates that B- and T-cell responses contribute to protective immunity against herpes virus infection. Glycoprotein B is a transmembrane envelope component of HSV-1 and HSV-2, which plays an important role in virion morphogenesis and penetration into host cells, and can induce neutralizing antibodies and protective T-cell response when it is used to immunize humans and animals. However, little is known about gB epitopes that are involved in B- and T-cell activities in vitro and in vivo. Thus, the HSV-2 gB sequence was screened using B- and T-cell epitope prediction systems, and the B-cell regions and the HLA-A*0201-restricted epitopes were identified. These B-cell epitopes elicited high IgG antibody titers in Balb/C mice, with a predominantly IgG1 subclass distribution, which indicated a Th2 bias. Specific IgGs induced by these two epitopes were evaluated as the neutralizing antibodies for virus neutralization. The predicted T-cell epitopes stabilized the HLA-A*0201 molecules on T(2) cells, and stimulate interferon-γ-secreting and cytotoxic CD8(+) T cells. Immunization with the predicted peptides reduced virus shedding and protected against lethal viral challenge in mice. The functional epitopes described herein, both B- and T-cell epitopes, are potentially implicated in vaccine development.     

Adenovirus particles that display the Plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutralizing antibodies in mice

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world.     

Immunization with different formulations of Mycobacterium tuberculosis antigen 85A induces immune responses with different specificity and protective efficacy

To test the relative efficacy of CD4 and CD8T cells in mediating protective immunity to Mycobacterium tuberculosis (Mtb), we compared three immunization regimes designed to induce preferentially each subset. BALB/c mice were immunized intranasally (i.n.) or parenterally with antigen 85A either in a recombinant Adenoviral vector (Ad85A), as recombinant protein (r85A) or as a set of overlapping 15mer peptides (p85A). For the first time we show that i.n. immunization with overlapping 85A synthetic peptides as well as Ad85A or r85A can provide protection against Mtb challenge. For all forms of the antigen, i.n. induces greater protection against Mtb challenge than parenteral immunization. Ad85A induces a predominantly CD8T cell response against the 85A_70–78 epitope, r85A a CD4 response to 85A_99–118 and p85A a balanced CD4/CD8 response to the CD4 85A_99–118 and CD8 85A_145–152 epitopes. Immune responses to CD4 85A_99–118 and CD8 85A_70–78 but not CD8 85A_145–152 are protective. Although Ad85A induces a strong response to the protective CD8 85A_70–78 epitope, we could not induce any response to this epitope by peptide immunization. These results show that although peptide immunization can induce protective immunity to Mtb challenge, it can also induce a response to a non-protective epitope in antigen 85A, indicating that the specificity of an immune response may be more important for protection against Mtb than its magnitude. These findings have important implications for the application of such vaccines in humans.     

Influenza virus CTL epitopes,remarkably conserved and remarkably variable

Virus-specific cytotoxic T lymphocytes (CTL) contribute to the control of virus infections including those caused by influenza viruses. Especially under circumstances when antibodies induced by previous infection or vaccination fail to recognize and neutralize the virus adequately, CTL are important and contribute to protective immunity. During epidemic outbreaks caused by antigenic drift variants and during pandemic outbreaks of influenza, humoral immunity against influenza viruses is inadequate. Under these circumstances, pre-existing CTL directed to the relatively conserved internal proteins of the virus may provide cross-protective immunity. Indeed, most of the known human influenza virus CTL epitopes are conserved. However, during the evolution of influenza A/H3N2 viruses, the most important cause of seasonal influenza outbreaks, variation in CTL epitopes has been observed. The observed amino acid substitutions affected recognition by virus-specific CTL and the human virus-specific CTL response in vitro. Examples of variable epitopes and their HLA restrictions are: NP_383–391/HLA-B*2705, NP_380–388/HLA-B*0801, NP_418–426/HLA-B*3501, NP_251–259/HLA-B*4002, NP_103–111/HLA-B*1503. In some cases amino acid substitutions occurred at anchor residues and in other cases at T cell receptor contact residues. It is of special interest that the R384G substitution in the NP_383–391 epitope was detrimental to virus fitness and was only tolerated in the presence of multiple functionally compensating co-mutations. In contrast, other epitopes, like the HLA-A*0201 restricted epitope from the matrix protein, M1_58–66, are highly conserved despite their immunodominant nature and the high prevalence of HLA-A*0201 in the population. A mutational analysis of this epitope indicated that it is under functional constraints. Also in influenza A viruses of other subtypes, including H5N1, the M1_58–66 is highly conserved.     

Evaluation of a prime-boost vaccine schedule with distinct adenovirus vectors against malaria in rhesus monkeys

A vaccine that elicits both specific antibodies and IFN-γ-producing T cells is required to protect against pre-erythrocytic malaria. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regimens, in particular ones containing live viral vector. We have demonstrated previously that adenovectors serotype 35 (Ads35) encoding the circumsporozoite (CS) antigen or liver-stage antigen-1 (LSA-1) are highly effective in improving the T-cell responses induced by immunizations with protein-based vaccines in a heterologous prime-boost schedule. Here we evaluated the potential of a heterologous prime-boost vaccination that combines the Ad35.CS vector with the serologically distinct adenovector Ad5.CS, in rhesus macaques, after establishing the potency in mice. We show that the heterologous Ad35.CS/Ad5.CS prime-boost regimen elicits both antibody responses and robust IFN-γ-producing CD8⁺ T-cell responses against the CS antigen. Analysis of the quality of the antibody responses in rhesus macaques, using indirect immunofluorescence assay (IFA) with Plasmodium falciparum-coated slides, demonstrated that this heterologous prime-boost regimen elicits a high titer of antibodies that are able to bind to P. falciparum sporozoites. Level of the IFA response was superior to the response measured with sera of an adult human population living in endemic malaria region. In conclusion, the combination of Ad35.CS, a vaccine based on a rare serotype adenovirus, with Ad5.CS or possibly another adenovector of a distinct serotype, induces a complex immune response that is required for protection against malaria, and is thus a highly promising approach for pediatric vaccination.     

Characterization of murine T-cell epitopes on mycobacterial DNA-binding protein 1 (MDP1) using DNA vaccination

Mycobacterial DNA-binding protein 1 (MDP1) is a major protein antigen in mycobacteria and induces protective immunity against Mycobacterium tuberculosis infection in mice. In this study we determined murine T-cell epitopes on MDP1 with MDP1 DNA immunization in mice. We analyzed interferon-γ production from the MDP1 DNA-immune splenocytes in response to 20-mer overlapping peptides covering MDP1 protein. We identified several CD4+ T-cell epitopes in three inbred mouse strains and one CD8+ T-cell epitope in C57BL/6 mice. These T-cell epitopes would be feasible for analysis of the role of MDP1-specific T-cells in protective immunity and for future vaccine design against M. tuberculosis infection.     

A single amino acid substitution in a recombinant G protein vaccine drastically curtails protective immunity against respiratory syncytial virus (RSV)

Recent studies have indicated a dominant T cell epitope located approximately between amino acids 184 and 203 on the respiratory syncytial virus (RSV) G protein. Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128-229) of the RSV G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids on vaccine efficacy. By changing two amino acids Arg 188 and Lys 192 to alanine, the ability of the Trx-G 128-229 fusion protein to protect mice against RSV challenge was virtually abolished. Mice immunized with the double mutant protein showed low levels of neutralizing antibodies and no pulmonary eosinophilic infiltrate, in contrast to that observed in mice immunized with wild type protein prior to RSV challenge. While less effective than the double mutant, mutation of either Arg 188 or Lys 192 to Ala drastically impaired the ability of immunized Trx-G 128-229 to induce neutralizing antibodies and to elicit pulmonary eosinophilia associated with RSV challenge. Despite low levels of virus-neutralizing antibodies, G protein-specific antibodies were detected by Western blotting in the sera from mice immunized with either of the single mutants (Arg 188 or Lys 192) but not the double mutant. Finally, immunization of mice with truncated forms of the Trx-G protein, showed partial protection against RSV challenge with Trx-G 128-188 but not with Trx-G 189-229. Taken together, the results indicate an important role for Arg 188 and Lys 192 in the induction of protective immunity and priming for eosinophilia against RSV. Furthermore, while the dominant protective linear epitope on the RSV G protein requires an intact sequence around Arg 188, there are additional, but less potent, protective epitopes upstream of Arg 188.     

Immunization with the RTS,S/AS malaria vaccine induces IFN-γ(+)CD4 T cells that recognize only discrete regions of the circumsporozoite protein and these specificities are maintained following booster immunizations and challenge

In a Phase 2a trial of the RTS,S/AS vaccine, we described significant association between protection against infection and vaccine-induced CD4 T cells. To determine whether processing of the circumsporozoite protein as a component of the RTS,S particulate antigen yields the same HLA-DR-restricted epitopes as those recognized by CD4 T cells from donors immunized by exposure to attenuated or infectious sporozoites we mapped the specificities of the RTS,S primed CD4 T cells by measuring IFN-γ cultured Elispot responses to pairs of overlapping 15 a.a. peptides that span the protein's C-terminus. Peptide pairs representing the previously described TH2R, T* and CS.T3 epitopes, were immunoprevalent and immunodominant. There was no response to the peptides corresponding to the human thrombospondin homology region. Responses to the CD4 T cell epitopes were restricted by multiple HLA-DR haplotypes. Of note, HLA-DR4 and HLA-DR11 restricted epitopes in the T* region and in the location on the CS protein defined by peptide pair 4, respectively. We conclude that processing of the CS protein derived from the RTS,S antigen leads to the generation of HLA-DR-restricted epitopes that are similar to those identified previously using CD4 T cells from subjects immunized with and protected by attenuated sporozoites or exposed to infectious sporozoites. This may in part account for the protective efficacy of the RTS,S/AS vaccine.     

Epitope-based sieve analysis of Plasmodium falciparum sequences from a FMP2.1/AS02A vaccine trial is consistent with differential vaccine efficacy against immunologically relevant AMA1 variants

To prevent premature dismissal of promising vaccine programs, it is critical to determine if lack of efficacy in the field is due to allele specific-efficacy, rather than to the lack of immunogenicity of the candidate antigen. Here we use samples collected during a field trial of the AMA1-based FMP2.1/AS02_A malaria vaccine, which incorporates the AMA1 variant encoded by the reference Plasmodium falciparum 3D7 strain, to assess the usefulness of epitope-based sieve analysis for the detection of vaccine-induced allele-specific immune responses.The samples used are from volunteers who received the malaria vaccine FMP2.1/AS02_A or a control (rabies vaccine), during a vaccine efficacy field trial, and who later developed malaria. In a previous study, P. falciparum DNA was extracted from all samples, and the ama1 locus amplified and sequenced. Here, a sieve analysis was used to measure T and B-cell escape, and difference in 3D7-like epitopes in the two treatment arms.Overall, no difference was observed in mean amino acid distance to the 3D7 AMA1 variant between sequences from vaccinees and controls in B-cell epitopes. However, we found a significantly greater proportion of 3D7-like T-cell epitopes that map to the AMA1 cluster one loop (c1L) region in the control vs. the vaccinee group (p = 0.02), consistent with allele-specific vaccine efficacy. Interestingly, AMA1 epitopes in infections from vaccinees had higher mean IC50, and consequently lower binding affinity, than epitopes generated from the control group (p = 0.01), suggesting that vaccine-induced selection impacted the immunological profile of the strains that pass through the sieve imposed by the vaccine-induced protection. These findings are consistent with a vaccine-derived sieve effect on the c1L region of AMA1 and suggest that sieve analyses of malaria vaccine trial samples targeted to epitopes identified in silico can help identify protective malaria antigens that may be efficacious if combined in a multivalent vaccine.     

Cell mediated immune response after challenge in Omp25 liposome immunized mice contributes to protection against virulent Brucella abortus 544

Brucellosis is a disease affecting various domestic and wild life species, and is caused by a bacterium Brucella. Keeping in view the serious economic and medical consequences of brucellosis, efforts have been made to prevent the infection through the use of vaccines. Cell-mediated immune responses [CMI] involving interferon gamma and cytotoxic CD4⁺ and CD8⁺ T cells are required for removal of intracellular Brucella. Omp25 has been reported to be involved in virulence of Brucella melitensis, Brucella abortus and Brucella ovis. In our previous study, we have shown the protective efficacy of recombinant Omp25, when administered intradermally. In this study, the recombinant Omp25 was formulated in PC–PE liposomes and PLGA microparticles, to enhance the protective immunity generated by it. Significant protection was seen with prime and booster liposome immunization in Balb/c mice against virulent B. abortus 544 as it was comparable to B. abortus S-19 vaccine strain. However, microparticle prime and booster immunization failed to give better protection when compared to B. abortus S-19 vaccine strain. This difference can be attributed to the stimulation of cell mediated immune response in PC–PE liposome immunized mice even after challenge which converted to cytotoxicity seen in CD4⁺ and CD8⁺ enriched lymphocytes. However, in PLGA microparticle immunized mice, cell mediated immunity was not generated after challenge as observed by decreased cytotoxicity of CD4⁺ and CD8⁺ enriched lymphocytes. Our study emphasizes on the importance of liposome encapsulating Omp25 immunization in conferring protection against B. abortus 544 challenge in Balb/c mice with a single dose immunization regimen.     

Addition of C3d-P28 adjuvant to a rabies DNA vaccine encoding the G5 linear epitope enhances the humoral immune response and confers protection

Rabies DNA vaccines based on full-length glycoprotein (G) induce virus neutralizing antibody (VNA) responses and protect against the virus challenge. Although conformational epitopes of G are the main target of VNAs, some studies have shown that a polypeptide linear epitope G5 is also able to induce VNAs. However, a G5 DNA vaccine has not been explored. While multiple doses of DNA vaccines are required in order to confer a protective immune response, this could be overcome by the inclusion of C3d-P28, a molecular adjuvant is know to improve the antibody response in several anti-viral vaccine models. To induce and enhance the immune response against rabies in mice, we evaluated two DNA vaccines based on the linear epitope G5 of Rabies Virus (RABV) glycoprotein (pVaxG5 vaccine) and another vaccine consisting of G5 fused to the molecular adjuvant C3d-P28 (pVaxF1 vaccine). VNA responses were measured in mice immunized with both vaccines. The VNA levels from the group immunized with pVaxG5 decreased gradually, while those from the group vaccinated with pVaxF1 remained high throughout the experimental study. After challenge with 22 LD50 of the Challenge Virus Strain (CVS), the survival rate of mice immunized with pVaxG5 and pVaxF1 was increased by 27% and 50% respectively, in comparison to the PBS group. Furthermore, the in vitro proliferation of anti-rabies specific spleen CD4+ and CD8+ T cells from mice immunized with pVaxF1 was observed. Collectively, these results suggest that the linear G5 epitope is a potential candidate vaccine. Furthermore, the addition of a C3d-P28 adjuvant contributed to enhanced protection, the sustained production of VNAs, and a specific T-cell proliferative response.     

Identification of non-CSP antigens bearing CD8 epitopes in mice immunized with irradiated sporozoites

Immunization of BALB/c mice with irradiated sporozoites (IrSp) of Plasmodium yoelii can lead to sterile immunity. The circumsporozoite protein (CSP) plays a dominant role in protection. Nevertheless after hyper-immunization with IrSp, complete protection is obtained in CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce antibodies [CSP-Tg/JhT(−/−)]. This protection is mediated exclusively by CD8⁺ T cells [1]. To identify the non-CSP protective T cell antigens, we studied the properties of 34 P. yoelii sporozoite antigens that are predicted to be secreted and to contain strong Kd-restricted CD8⁺ T cell epitopes. The synthetic peptides corresponding to the epitopes were used to screen for the presence of peptide-specific CD8⁺ T cells secreting interferon-γ (IFN-γ) in splenocytes from CSP-Tg/JhT(−/−) BALB/c mice hyper immunized with IrSp. However, the numbers of IFN-γ-secreting splenocytes specific for the non-CSP antigen-derived peptides were 20-100 times lower than those specific for the CSP-specific peptide. When mice were immunized with recombinant adenoviruses expressing selected non-CSP antigens, the animals were not protected against challenge with P. yoelii sporozoites although large numbers of CD8⁺ specific T cells were generated.     

Immunogenicity of Ty-VLP bearing a CD8(+) T cell epitope of the CS protein of P. yoelii: enhanced memory response by boosting with recombinant vaccinia virus

We characterized the immunogenicity of the hybrid Ty-virus-like carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein of Plasmodium yoelii (TyCS-VLP), a rodent malaria parasite. Balb/c mice were immunized with hybrid TyCS-VLP, and their CS-specific CD8(+) T cell response was quantitatively evaluated with the ELISPOT assay, based on the enumeration of epitope specific gamma-interferon secreting CD8(+) T cell. A single immunization with the TyCS-VLP by a variety of routes and doses indicated that the maximal response occurred in mice, which were immunized with 50 micrograms of these particles, administered via intramuscular. Combined immunization of mice with this TyCS-VLP followed by recombinant vaccinia virus expressing the entire P. yoelii CS protein (VacPyCS) or irradiated sporozoites, induced high levels of IFN-gamma-producing cells. The immunization regime, priming with TyCS-VLP and boosting with VacPyCS generated a potent protective immune response, which strongly inhibited P. yoelii liver stages development and protected 62% of the mice against a subsequent live P. yoelii sporozoite challenge.     

Natural selection maintains a stable polymorphism at the circumsporozoite protein locus of Plasmodium falciparum in a low endemic area

Circumsporozoite protein gene sequences of Plasmodium falciparum were collected in 1996–1997 and in 2006–2007 from a single endemic area in Thailand. Repeat units were more similar within the same haplotype than between haplotypes, supporting the hypothesis that repeat arrays evolve by a process of concerted evolution. There was evidence that natural selection has favored amino acid changes in the Th2R and Th3R T-cell epitope regions. One haplotype in these epitopes, designated *5/*1, occurred in approximately 70% of sequences in both collection periods. The most common other haplotypes differed from *5/*1 by at least two amino acid replacements; and divergence in the epitopes was correlated with divergence in the repeats. These patterns are most consistent with balancing selection driven by interactions with the immune system of the vertebrate host, probably involving both T-cell recognition of the Th2R and Th3R epitopes and antibody responses to the repeats.