Leading Vaccine Candidate Research Articles

Plasmodium vivax: comparison of immunogenicity among proteins expressed in the cell-free systems of Escherichia coli and wheat germ by suspension array assays

BackgroundIn vitro cell-free systems for protein expression with extracts from prokaryotic (Escherichia coli) or eukaryotic (wheat germ) cells coupled to solid matrices have offered a valid approach for antigen discovery in malaria research. However, no comparative analysis of both systems is presently available nor the usage of suspension array technologies, which offer nearly solution phase kinetics.MethodsFive Plasmodium vivax antigens representing leading vaccine candidates were expressed in the E. coli and wheat germ cell-free systems at a 50 μl scale. Products were affinity purified in a single-step and coupled to luminex beads to measure antibody reactivity of human immune sera.ResultsBoth systems readily produced detectable proteins; proteins produced in wheat germ, however, were mostly soluble and intact as opposed to proteins produced in E. coli, which remained mostly insoluble and highly degraded. Noticeably, wheat germ proteins were recognized in significantly higher numbers by sera of P. vivax patients than identical proteins produced in E. coli.ConclusionsThe wheat germ cell-free system offers the possibility of expressing soluble P. vivax proteins in a small-scale for antigen discovery and immuno-epidemiological studies using suspension array technology.

Transgene Optimization, Immunogenicity and In Vitro Efficacy of Viral Vectored Vaccines Expressing Two Alleles of Plasmodium falciparum AMA1

BackgroundApical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood-stage malaria, although to date numerous clinical trials using mainly protein-in-adjuvant vaccines have shown limited success. Here we describe the pre-clinical development and optimization of recombinant human and simian adenoviral (AdHu5 and ChAd63) and orthopoxviral (MVA) vectors encoding transgene inserts for Plasmodium falciparum AMA1 (PfAMA1).Methodology/Principal FindingsAdHu5-MVA prime-boost vaccination in mice and rabbits using these vectors encoding the 3D7 allele of PfAMA1 induced cellular immune responses as well as high-titer antibodies that showed growth inhibitory activity (GIA) against the homologous but not heterologous parasite strains. In an effort to overcome the issues of PfAMA1 antigenic polymorphism and pre-existing immunity to AdHu5, a simian adenoviral (ChAd63) vector and MVA encoding two alleles of PfAMA1 were developed. This antigen, composed of the 3D7 and FVO alleles of PfAMA1 fused in tandem and with expression driven by a single promoter, was optimized for antigen secretion and transmembrane expression. These bi-allelic PfAMA1 vaccines, when administered to mice and rabbits, demonstrated comparable immunogenicity to the mono-allelic vaccines and purified serum IgG now showed GIA against the two divergent strains of P. falciparum encoded in the vaccine. CD8+ and CD4+ T cell responses against epitopes that were both common and unique to the two alleles of PfAMA1 were also measured in mice.Conclusions/SignificanceOptimized transgene inserts encoding two divergent alleles of the same antigen can be successfully inserted into adeno- and pox-viral vaccine vectors. Adenovirus-MVA immunization leads to the induction of T cell responses common to both alleles, as well as functional antibody responses that are effective against both of the encoded strains of P. falciparum in vitro. These data support the further clinical development of these vaccine candidates in Phase I/IIa clinical trials.

The case for a subunit vaccine against malaria

New technologies and some disillusionment with subunit vaccines has led to increased interest in the development of whole parasite vaccines for malaria. Instead, the current priority should be to build on the partial success of the recombinant protein sporozoite vaccine, RTS,S. There are many possible options for delivering a subunit vaccine but the simplest option, formulating recombinant proteins in an adjuvant, should be fully explored. Numerous options exist for inducing heightened immune responses and for tackling the problem of diversity, but development of recombinant protein subunit vaccines requires a more detailed knowledge of the conformation of the leading vaccine candidates.

Genetic diversity of Plasmodium vivax Duffy Binding Protein II (PvDBPII) under unstable transmission and low intensity malaria in Sri Lanka

Elucidating the genetic diversity of the Duffy Binding Protein II (PvDBPII), a leading vaccine candidate for vivax malaria, in different geographical settings is vital. In Sri Lanka malaria transmission is unstable with low intensity. A relatively high level of allelic diversity, with 27 polymorphic nucleotides and 33 (aa) haplotypes was detected among the PvdbpII gene in 100 local Plasmodium vivax isolates collected from two hypoendemic areas, and from a non endemic area of the country. Mutations, recombination and balancing selection seem to maintain the observed local allelic diversity of PvdbpII. Lack of gene flow was evidenced by high Fst values between the two endemic study sites. Some of the aa polymorphisms may alter the binding and expression capacity of predicted T cell epitopes in PvDBPII. Of the 8 binding inhibitory linear B cell epitopes, 2 (H2 and M1) in the vicinity of the exact binding region of PvDBPII appeared to be highly conserved in Sri Lankan, Iran and Colombian isolates, while H3, M2, M3 and L3 neutralizing epitopes seem to be polymorphic globally, with H1 and L2 conserved in Colombian, South Korean and Iran isolates. In comparison to the reference Sal-1 strain, among 402 world-wide isolates (302 global and 100 local), 121 aa polymorphisms and 138 haplotypes were recorded of which 3 aa polymorphisms and 21 haplotypes seem to be unique to Sri Lanka. PvdbpII phylogeny suggests that local P. vivax parasites represent a sample of the global population. The ubiquitous presence of some PvDBPII aa haplotypes among both local and global P. vivax isolates may aid future vaccination strategies based on PvDBPII.

Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins

Gene coding for leucine aminopeptidase (LAP), a metalloprotease, was identified in the tropical liver fluke, Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopeptidase of the temperate liver fluke, Fasciola hepatica. The recombinant leucine aminopeptidase protein was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes was conducted with these two recombinant proteins, with 150 and 300μg of leucine aminopeptidase and a cocktail of 150μg each of recombinant leucine aminopeptidase and peroxiredoxin in three groups, respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization with these antigens. A challenge study with 400 metacercariae did not show a significant protection in terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate vaccine molecule. Identification of leucine aminopeptidase gene and evaluation of the protein for its protective efficacy in buffaloes is the first scientific report on this protein in F. gigantica.

Characterization of Conserved Combined T and B Cell Epitopes in Leptospira interrogansMajor Outer Membrane Proteins OmpL1 and LipL41

BackgroundLeptospira interrogans are bacterial pathogens of animal that cause zoonotic infections in human. Outer membrane proteins of leptospire are among the most effective antigens which can stimulate remarkable immune responses during the infection processes, and thus are currently considered leading candidate vaccine antigens. The objective of the present study is to predict and confirm major combined B and T cell epitopes of leptospiral outer membrane proteins OmpL1 and LipL41, as well as to evaluate their capacity in the induction of immune responses in BALB/c mice.ResultsIn this study, four epitopes from OmpL1 and four from LipL41 conserved regions were evaluated for their potential utilization in leptospire vaccines. Firstly, combined B and T cell epitopes were predicted by softwares and expressed using a phage display system. OmpL1 residues 87-98 and 173-191 (OmpL187-98 and OmpL1173-191) and LipL4130-48, LipL41233-256 of LipL41 were identified as immunodominant B cell epitopes by Western blot. Epitopes OmpL1173-191, OmpL1297-320 of OmpL1 and LipL41233-256, LipL41263-282 of LipL41 were identified as immunodominant CD4+ T cell epitopes through proliferation analysis of splenocytes from recombinant OmpL1 (rOmpL1) or recombinant LipL41 (rLipL41)-immunized BALB/c (H-2d) mice. These epitopes induced responses of CD4+ T cells and Th1 (T helper cells) type cytokine responses during the infection.ConclusionThis work identified combined T and B cell immunodominant epitopes in outer membrane proteins OmpL1 and LipL41 of Leptospira interrogans. OmpL1173-191 of OmpL1 and LipL41233-256 of LipL41 could be useful in a vaccine against Leptospira. The findings could also contribute to the development of effective cross-protective vaccine strategies for leptospirosis.

Limited Polymorphism of the Plasmodium vivax Merozoite Surface Protein 1 Gene in Isolates from Turkey

The 200-kD merozoite surface protein of Plasmodium vivax (PvMSP-1) is one of the leading vaccine candidates against P. vivax malaria. However, the gene encoding PvMSP-1 (pvmsp1) is highly polymorphic and is a major obstacle to effective vaccine development. To further understand polymorphism in pvmsp1, we obtained 30 full-length pvmsp1 sequences from southeastern Turkey. Comparative analysis of sequences from Turkey and other areas showed substantially limited polymorphism. Substitutions were found at 280 and 162 amino acid sites in samples from other regions and those from Turkey, respectively. Eight substitutions were unique to Turkey. In one of them, D/E at position 1706 in the C-terminal 19-kD region, the K/E change at 1709 was the only polymorphism previously known. Limited diversity was also observed in microsatellites. Data suggest a recent population bottleneck in Turkey that may have obscured a signature for balancing selection in the C-terminal 42-kD region, which was otherwise detectable in other areas.

New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1.

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

Immunoglobulin GM 3 23 5,13,14 phenotype is strongly associated with IgG1 antibody responses to Plasmodium vivax vaccine candidate antigens PvMSP1-19 and PvAMA-1

BackgroundHumoral immune responses play a key role in the development of immunity to malaria, but the host genetic factors that contribute to the naturally occurring immune responses to malarial antigens are not completely understood. The aim of the present investigation was to determine whether, in subjects exposed to malaria, GM and KM allotypes--genetic markers of immunoglobulin γ and κ-type light chains, respectively--contribute to the magnitude of natural antibody responses to target antigens that are leading vaccine candidates for protection against Plasmodium vivax.MethodsSera from 210 adults, who had been exposed to malaria transmission in the Brazilian Amazon endemic area, were allotyped for several GM and KM determinants by a standard hemagglutination-inhibition method. IgG subclass antibodies to P. vivax apical membrane antigen 1 (PvAMA-1) and merozoite surface protein 1 (PvMSP1-19) were determined by an enzyme-linked immunosorbent assay. Multiple linear regression models and the non-parametric Mann-Whitney test were used for data analyses.ResultsIgG1 antibody levels to both PvMSP1-19 and PvAMA-1 antigens were significantly higher (P = 0.004, P = 0.002, respectively) in subjects with the GM 3 23 5,13,14 phenotype than in those who lacked this phenotype.ConclusionsResults presented here show that immunoglobulin GM allotypes contribute to the natural antibody responses to P. vivax malaria antigens. These findings have important implications for the effectiveness of vaccines containing PvAMA-1 or PvMSP1-19 antigens. They also shed light on the possible role of malaria as one of the evolutionary selective forces that may have contributed to the maintenance of the extensive polymorphism at the GM loci.

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.

The meningococcal vaccine candidate Neisserial surface protein A (NspA) binds to Factor H and enhances meningococcal resistance to complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.

Identification of key residues involved in fibril formation by the conserved N-terminal region of Plasmodium falciparum merozoite surface protein 2 (MSP2)

Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. MSP2 is assumed to have a role in erythrocyte invasion and is a leading vaccine candidate. Recombinant MSP2 forms amyloid-like fibrils upon storage, as do peptides corresponding to sequences in the conserved N-terminal region, which constitutes the structural core of fibrils formed by full-length MSP2. We have investigated the roles of individual residues in fibril formation and local ordered structure in two peptides, a recombinant 25-residue peptide corresponding to the entire N-terminal domain of mature MSP2 and an 8-residue peptide from the central region of this domain (residues 8–15). Both peptides formed fibrils that were similar to amyloid-like fibrils formed by full-length MSP2. Phe11 and Ile12 have important roles both in stabilising local structure in these peptides and promoting fibril formation; the F11A and I12A mutants of MSP2 8–15 were essentially unstructured in solution and fibril formation at pH 7.4 and 4.7 was markedly retarded. The T10A mutant showed intermediate behaviour, having a less well defined structure than wild-type and slower fibril formation at pH 7.4. The mutation of Phe11 and Ile12 in MSP2 1–25 significantly retarded but did not abolish fibril formation, indicating that these residues also play a key role in fibril formation by the entire N-terminal conserved region. These mutations had little effect on the aggregation of full-length MSP2, however, suggesting that regions outside the conserved N-terminus have unanticipated importance for fibril formation in the full-length protein.

Allele frequency-based and polymorphism-versus-divergence indices of balancing selection in a new filtered set of polymorphic genes in Plasmodium falciparum.

Signatures of balancing selection operating on specific gene loci in endemic pathogens can identify candidate targets of naturally acquired immunity. In malaria parasites, several leading vaccine candidates convincingly show such signatures when subjected to several tests of neutrality, but the discovery of new targets affected by selection to a similar extent has been slow. A small minority of all genes are under such selection, as indicated by a recent study of 26 Plasmodium falciparum merozoite-stage genes that were not previously prioritized as vaccine candidates, of which only one (locus PF10_0348) showed a strong signature. Therefore, to focus discovery efforts on genes that are polymorphic, we scanned all available shotgun genome sequence data from laboratory lines of P. falciparum and chose six loci with more than five single nucleotide polymorphisms per kilobase (including PF10_0348) for in-depth frequency–based analyses in a Kenyan population (allele sample sizes >50 for each locus) and comparison of Hudson–Kreitman–Aguade (HKA) ratios of population diversity (π) to interspecific divergence (K) from the chimpanzee parasite Plasmodium reichenowi. Three of these (the msp3/6-like genes PF10_0348 and PF10_0355 and the surf4.1 gene PFD1160w) showed exceptionally high positive values of Tajima's D and Fu and Li's F indices and have the highest HKA ratios, indicating that they are under balancing selection and should be prioritized for studies of their protein products as candidate targets of immunity. Combined with earlier results, there is now strong evidence that high HKA ratio (as well as the frequency-independent ratio of Watterson's θ/K) is predictive of high values of Tajima's D. Thus, the former offers value for use in genome-wide screening when numbers of genome sequences within a species are low or in combination with Tajima's D as a 2D test on large population genomic samples.

Prolonged Antigen Presentation Is Required for Optimal CD8+ T Cell Responses against Malaria Liver Stage Parasites

Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization—a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites (137.16)

Abstract Immunization with irradiated sporozoites is the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization - a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate into liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite antigen.

The Relationship between Anti-merozoite Antibodies and Incidence of Plasmodium falciparum Malaria: A Systematic Review and Meta-analysis

One of the criteria to objectively prioritize merozoite antigens for malaria vaccine development is the demonstration that naturally acquired antibodies are associated with protection from malaria. However, published evidence of the protective effect of these antibodies is conflicting. We performed a systematic review with meta-analysis of prospective cohort studies examining the association between anti-merozoite immunoglobin (Ig) G responses and incidence of Plasmodium falciparum malaria. Two independent researchers searched six databases and identified 33 studies that met predefined inclusion and quality criteria, including a rigorous definition of symptomatic malaria. We found that only five studies were performed outside sub-Saharan Africa and that there was a deficiency in studies investigating antibodies to leading vaccine candidates merozoite surface protein (MSP)-1(42) and erythrocyte binding antigen (EBA)-175. Meta-analyses of most-studied antigens were conducted to obtain summary estimates of the association between antibodies and incidence of P. falciparum malaria. The largest effect was observed with IgG to MSP-3 C terminus and MSP-1(19) (responders versus nonresponders, 54%, 95% confidence interval [CI] [33%-68%] and 18% [4%-30%] relative reduction in risk, respectively) and there was evidence of a dose-response relationship. A tendency towards protective risk ratios (RR<1) was also observed for individual study estimates for apical membrane antigen (AMA)-1 and glutamate-rich protein (GLURP)-R0. Pooled estimates showed limited evidence of a protective effect for antibodies to MSP-1 N-terminal regions or MSP-1-EGF (epidermal growth factor-like modules). There was no significant evidence for the protective effect for MSP-2 (responders versus nonresponders pooled RR, MSP-2(FC27) 0.82, 95% CI 0.62-1.08, p = 0.16 and MSP-2(3D7) 0.92, 95% CI 0.75-1.13, p = 0.43). Heterogeneity, in terms of clinical and methodological diversity between studies, was an important issue in the meta-analysis of IgG responses to merozoite antigens. These findings are valuable for advancing vaccine development by providing evidence supporting merozoite antigens as targets of protective immunity in humans, and to help identify antigens that confer protection from malaria. Further prospective cohort studies that include a larger number of lead antigens and populations outside Africa are greatly needed to ensure generalizability of results. The reporting of results needs to be standardized to maximize comparability of studies. We therefore propose a set of guidelines to facilitate the uniform reporting of malaria immuno-epidemiology observational studies. Please see later in the article for the Editors' Summary.

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1(19)) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1(19) in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1(19). The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1(19) in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1(19). It was observed that the higher expression level of PfMSP-1(19) was essentially associated with the generation of higher level of incorrectly folded PfMSP-1(19). A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1(19) from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1(19) suggested that it was highly pure, homogeneous, and correctly folded.

Plasmodium falciparum apical membrane antigen 1 vaccine elicits multifunctional CD4 cytokine-producing and memory T cells

The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading vaccine candidate and was tested for safety and immunogenicity in human Phase I Clinical Trials. PBMC from vaccine recipients were analyzed by flow cytometric methods to determine the nature of T-cell responses and AMA1-reactive memory T cells. Both CD4 and CD8 T cells produced a number of cytokines following AMA1 re-stimulation, with IL-5-producing cells at the highest frequency, consistent with a Th2 bias. The relative frequency of multifunctional cells synthesizing Th1 cytokines IFN-γ, IL-2 and TNF-α changed after each vaccination. Interestingly, median fluorescence intensity measurements revealed that cells producing more than one cytokine contributed greater quantities of each cytokine than cell populations that produced each of the cytokines alone. AMA1 vaccination also elicited the development of memory cell populations, and both central and effector memory T cells were identified concurrently after the AMA1 vaccination. The detailed profile of multifunctional T-cell responses to AMA1 presented here will advance our ability to assess the immunogenicity of human malarial vaccines.

Moving forward: a mucosal vaccine against group A streptococcus

Streptococcus pyogenes, commonly referred to as group A streptococcus (GAS), colonizes the epithelial surfaces of the upper respiratory tract and the skin, where it causes a myriad of diseases that vary in clinical presentation and severity. Several candidate GAS vaccines are now approaching or have entered human clinical trials. However, the preclinical evaluation for the leading vaccine candidates has been premised on their ability to induce systemic protection through parenteral immunization. While systemic immunity has proven effective in preventing GAS dissemination and associated disease, it may not prove to be the optimal approach for inducing mucosal immunity against GAS. With this in mind, many researchers are moving toward testing the efficacy of their GAS vaccine candidate when delivered to the mucosal surface. This review discusses the interaction of GAS with the mucosal immune system and the approaches taken thus far in developing a mucosal GAS vaccine.

IgG subclasses pattern and high-avidity antibody to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in an unstable hypoendemic region in Iran

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1 19) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1 19 were evaluated in individuals ( n = 94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1 19 and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1 19, the frequency of antibodies was determined in the infected subjects ( n = 74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1 19 was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1 19. These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1 19-based vaccine.