Strain-specific Protective Immunity Research Articles

Strain-Specific Protective Effect of the Immunity Induced by Live Malarial Sporozoites under Chloroquine Cover

The efficacy of a whole-sporozoite malaria vaccine would partly be determined by the strain-specificity of the protective responses against malarial sporozoites and liver-stage parasites. Evidence from previous reports were inconsistent, where some studies have shown that the protective immunity induced by irradiated or live sporozoites in rodents or humans were cross-protective and in others strain-specific. In the present work, we have studied the strain-specificity of live sporozoite-induced immunity using two genetically and immunologically different strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkeys. Two groups of monkeys were immunized against live sporozoites of either the Pc746 (n = 5), or the PcCeylon (n = 4) strain, by the bites of 2–4 sporozoite-infected Anopheles tessellates mosquitoes per monkey under concurrent treatments with chloroquine and primaquine to abrogate detectable blood infections. Subsequently, a group of non-immunized monkeys (n = 4), and the two groups of immunized monkeys were challenged with a mixture of sporozoites of the two strains by the bites of 2–5 infective mosquitoes from each strain per monkey. In order to determine the strain-specificity of the protective immunity, the proportions of parasites of the two strains in the challenge infections were quantified using an allele quantification assay, Pyrosequencing™, based on a single nucleotide polymorphism (SNP) in the parasites’ circumsporozoite protein gene. The Pyrosequencing™ data showed that a significant reduction of parasites of the immunizing strain in each group of strain-specifically immunized monkeys had occurred, indicating a stronger killing effect on parasites of the immunizing strain. Thus, the protective immunity developed following a single, live sporozoite/chloroquine immunization, acted specifically against the immunizing strain and was, therefore, strain-specific. As our experiment does not allow us to determine the parasite stage at which the strain-specific protective immunity is directed, it is possible that the target of this immunity could be either the pre-erythrocytic stage, or the blood-stage, or both.

Strain specific protective immunity induced by pre-erythrocytic infection of toque monkeys with Plasmodium cynomolg

Strain specific protective immunity (SSPI) was investigated using two strains of Plasmodium cynomolgi, Pc746 and PcCeylon, in toque monkey. Two groups of monkeys were immunized against either Pc746 (n=5) or PcCeylon (n=4), by giving bites with 2-4 sporozoite-infected Anopheles tessellates mosquitoes per monkey. Primary blood infection was prevented by simultaneous chloroquine cover and secondary, hypnozoite-induced, blood infection was prevented by treating the monkeys one month later with primaquine. The two immunized groups and a group of unimmunized monkeys (n=4) were given a mixed-strain sporozoite challenge infection, 140 and 100 days respectively after immunization. Parasite DNA was collected for 5-8 consecutive days after parasitaemia reached 0.05% or above. The proportions of the two parasite strains in these samples were quantified using a PyrosequencingTM assay based on SNPs in MSP1 and CSP genes. In subsequent blood infections in immunized monkeys the earliest recorded proportion of parasites of an immunizing strain was significantly lower than its proportion in monkeys immunized against a heterologous strain (P=0.014 and 0.027 for the two SNPs) and the proportions of an immunizing strain tended to decline during the period of sample collection. These results show that a parasite strain specific protective immunity to P. cynomolgi was indcued following a sporozoite induced pre-erythrocytic infection. This immunity may have been directed against the liver stages or against the blood stage parasites, or against both

A single parasite gene determines strain-specific protective immunity against malaria: The role of the merozoite surface protein I

Despite many decades of research, no registered vaccine against the pathogenic blood stages of the malaria parasite exists, translating into the loss of many hundreds of thousands of young lives each year in tropical Africa. Although many parasite proteins have been shown to induce immune responses in the host, proof for their induction of protective immunity is still lacking. We previously reported a novel genetic approach called linkage group selection (LGS) for rapid identification of target antigens of strain-specific protective immunity (SSPI) against malaria. In preliminary LGS experiments, we crossed two genetically distinct strains of Plasmodium chabaudi chabaudi and subjected their progeny to selection in strain-specifically immunised mice, measuring the effects of SSPI selection with low coverage/resolution genetic markers. In the present study, through application of high coverage/resolution, single nucleotide polymorphism (SNP) markers spanning all 14 parasite chromosomes, we analysed 35 SSPI selection events on different populations of progeny parasites. Here we report a comprehensive high resolution genome-wide analysis of the effects of strain-specific immune selection on blood stage parasites. Our analyses consistently identify a single genomic region spanning ∼79 kb on chromosome 8 as the region controlling SSPI. Within this region, one gene (that of merozoite surface protein 1, MSP-1) accounted for >60% of genetic polymorphism and was most frequently under greatest reduction under SSPI. These results, combined with those of an independent LGS analysis of a different genetic cross with different parental strains, demonstrate that more than any other locus, the gene for MSP-1 determines the effect of strain-specific protective immunity against malaria in these host-parasite combinations. Our results provide unique insight into the precise timing of the parasite killing immune response against progeny parasites carrying specific alleles of MSP-1; these findings pave the way for investigating which part(s) of this highly polymorphic molecule mediate the protective immune response.

Strain-specific protective immunity following vaccination against experimental Trypanosoma cruzi infection

Immunisation with Amastigote Surface Protein 2 ( asp-2) and trans-sialidase ( ts) genes induces protective immunity in highly susceptible A/Sn mice, against infection with parasites of the Y strain of Trypanosoma cruzi. Based on immunological and biological strain variations in T. cruzi parasites, our goal was to validate our vaccination results using different parasite strains. Due to the importance of the CD8 + T cells in protective immunity, we initially determined which strains expressed the immunodominant H-2K k-restricted epitope TEWETGQI. We tested eight strains, four of which elicited immune responses to this epitope (Y, G, Colombian and Colombia). We selected the Colombian and Colombia strains for our studies. A/Sn mice were immunised with different regimens using both T. cruzi genes ( asp-2 and ts) simultaneously and subsequently challenged with blood trypomastigotes. Immune responses before the challenge were confirmed by the presence of specific antibodies and peptide-specific T cells. Genetic vaccination did not confer protective immunity against acute infection with a lethal dose of the Colombian strain. In contrast, we observed a drastic reduction in parasitemia and a significant increase in survival, following challenge with an otherwise lethal dose of the Colombia strain. In many surviving animals with late-stage chronic infection, we observed alterations in the heart's electrical conductivity, compared to naive mice. In summary, we concluded that immunity against T. cruzi antigens, similar to viruses and bacteria, may be strain-specific and have a negative impact on vaccine development.

Limited global diversity of the Plasmodium vivax merozoite surface protein 4 gene

Merozoite surface proteins (MSPs) of the malaria parasites are major candidates for vaccine development targeting asexual blood stages. However, the diverse antigenic repertoire of these antigens that induce strain-specific protective immunity in human is a major challenge for vaccine design and often determines the efficacy of a vaccine. Here we further assessed the genetic diversity of Plasmodium vivax MSP4 (PvMSP4) protein using 195 parasite samples collected mostly from Thailand, Indonesia and Brazil. Overall, PvMSP4 is highly conserved with only eight amino acid substitutions. The majority of the haplotype diversity was restricted to the two short tetrapeptide repeat arrays in exon 1 and 2, respectively. Selection and neutrality tests indicated that exon 1 and the entire coding region of PvMSP4 were under purifying selection. Despite the limited nucleotide polymorphism of PvMSP4, significant genetic differentiation among the three major parasite populations was detected. Moreover, microgeographical heterogeneity was also evident in the parasite populations from different endemic areas of Thailand.

Strain-specific immunity may drive adaptive polymorphism in the merozoite surface protein 1 of the rodent malaria parasite Plasmodium chabaudi

Clinical immunity against malaria is slow to develop, poorly understood and strongly strain-specific. Understanding how strain-specific immunity develops and identifying the parasite antigens involved is crucial to developing effective vaccines against the disease. In previous experiments we have shown that strain-specific protective immunity (SSPI) exists between genetically distinct strains (cloned lines) of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice [Cheesman, S., Raza, A., Carter, R., 2006. Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite P. chabaudi chabaudi in mice. Infect. Immun. 74, 2996-3001]. In two subsequent studies, we identified the highly polymorphic Merozoite Surface Protein 1 (MSP-1) as being the principal candidate molecule for the control of SSPI against P. c. chabaudi malaria [Martinelli et al., 2005; Pattaradilokrat, S., Cheesman, S.J., Carter R., 2007. Linkage group selection: towards identifying genes controlling strain-specific protective immunity in malaria. PLoS ONE 2(9):e857]. In the present study, we sequenced the whole msp1 gene of several genetically distinct strains of P. chabaudi and found high levels of genetic diversity. Protein sequence alignments reveal extensive allelic polymorphism between the P. chabaudi strains, concentrated primarily within five regions of the protein. The 3′-end sequence region, encoding the C-terminal 21kDa region (MSP-121), which is analogous and homologous to MSP-119 of Plasmodium falciparum, appears to have been subject to balancing selection. We have found that the strains with the lowest sequence identity at MSP-121 (i.e. AS/CB and AJ/CB) induce robust and reciprocal SSPI in experimental mice. In contrast, two strains that do not induce reciprocal SSPI are identical at the 21kDa region. Final identification of the region(s) controlling SSPI will provide important information to help guide decisions about MSP-1 based vaccines.

Evidence for strain‐specific protective immunity against blood‐stage parasites of Plasmodium cynomolgi in toque monkey

We have conducted experiments to test the induction of strain-specific protective immunity against Plasmodium cynomolgi infections in toque monkeys. Plasmodium cynomolgi is closely related biologically and genetically to the human malaria parasite, P. vivax. Two groups of monkeys were immunized against either of two strains of P. cynomolgi, namely PcCeylon and Pc746, by giving two successive drug-cured infections with asexual blood-stage parasites of one or the other strain, 12-weeks apart. To test for strain-specific protective immunity these infection-immunized monkeys were challenged 8 weeks later with a mixture of asexual blood-stage parasites of both strains. A pyrosequencing-based assay was used to quantify the proportion of parasites that survived in the challenge infections. The assay was based on a SNP within the P. cynomolgi Merozoite Surface Protein-1 gene. Compared to their behaviour in nonimmunized monkeys, the growth of parasites of the homologous (immunizing) strain in mixed-strain challenge infections in the immunized monkeys were reduced relative to that of the nonimmunizing strain. These results indicate the development of blood infection-induced strain-specific protective immunity against P. cynomolgi in toque monkeys. The work prepares for using genetic analysis to identify target antigens of strain-specific protective immunity in this host and malaria parasite combination.

Linkage Group Selection: Towards Identifying Genes Controlling Strain Specific Protective Immunity in Malaria

Protective immunity against blood infections of malaria is partly specific to the genotype, or strain, of the parasites. The target antigens of Strain Specific Protective Immunity are expected, therefore, to be antigenically and genetically distinct in different lines of parasite. Here we describe the use of a genetic approach, Linkage Group Selection, to locate the target(s) of Strain Specific Protective Immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi. In a previous such analysis using the progeny of a genetic cross between P. c. chabaudi lines AS-pyr1 and CB, a location on P. c. chabaudi chromosome 8 containing the gene for merozoite surface protein-1, a known candidate antigen for Strain Specific Protective Immunity, was strongly selected. P. c. chabaudi apical membrane antigen-1, another candidate for Strain Specific Protective Immunity, could not have been evaluated in this cross as AS-pyr1 and CB are identical within the cell surface domain of this protein. Here we use Linkage Group Selection analysis of Strain Specific Protective Immunity in a cross between P. c. chabaudi lines CB-pyr10 and AJ, in which merozoite surface protein-1 and apical membrane antigen-1 are both genetically distinct. In this analysis strain specific immune selection acted strongly on the region of P. c. chabaudi chromosome 8 encoding merozoite surface protein-1 and, less strongly, on the P. c. chabaudi chromosome 9 region encoding apical membrane antigen-1. The evidence from these two independent studies indicates that Strain Specific Protective Immunity in P. c. chabaudi in mice is mainly determined by a narrow region of the P. c. chabaudi genome containing the gene for the P. c. chabaudi merozoite surface protein-1 protein. Other regions, including that containing the gene for P. c. chabaudi apical membrane antigen-1, may be more weakly associated with Strain Specific Protective Immunity in these parasites.

Strain-Specific Pulmonary Defense Achieved after Repeated Airway Immunizations with Non-Typeable Haemophilus Influenzae in a Mouse Model

Strain-specific immune responses may play a critical role in the acute exacerbation of chronic obstructive pulmonary disease (COPD) caused by Haemophilus influenzae (NTHi), and the outer membrane protein P2 is one of surface antigens of NTHi, which may contribute to the strain-specific protective immunity. We examined whether repeated airway immunizations with killed-NTHi strains bearing different P2 molecules were capable of inducing protective immunity against homologous or heterologous strains in the lungs of a mouse model. Three different strains of NTHi were used in this study. Three serial intratracheal (IT) immunizations of a single strain or three different strains of NTHi led to the production of cross-reactive immunoglobulins G and A in bronchoalveolar lavage fluids. Three serial IT immunizations with a single strain enhanced the bacterial clearance of the homologous strain in the lungs, but no enhancement of bacterial clearance was found with three serial IT immunizations of heterologous strains. The enhancement in bacterial clearance, therefore, appears to be primarily strain-specific. Enhanced bacterial clearance of a heterologous strain was also found after three serial IT immunizations of a single strain among two of the three strains employed for bacterial challenge. These findings suggest that P2 molecules and surface antigens other than P2 are involved in the development of pulmonary defense against NTHi in mice. Our data may explain, in part, why patients with COPD experience recurrent NTHi infections.

Merozoite Surface Antigen 2 Proteins of Babesia bovis Vaccine Breakthrough Isolates Contain a Unique Hypervariable Region Composed of Degenerate Repeats

The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes of vaccine breakthrough isolates would also encode a diverse set of proteins, the complete msa-2 locus was characterized from 12 Australian B. bovis strains and isolates, including two vaccine strains and eight vaccine breakthrough isolates, and compared to the loci in previously and newly characterized American strains. In contrast to American strains, the msa-2 loci of all Australian strains and isolates examined contain, in addition to msa-2c, only a solitary gene (designated msa-2a/b) closely related to American strain msa-2a and msa-2b. Nevertheless, the proteins encoded by these genes are quite diverse both between and within geographic regions and harbor evidence of genetic exchange among other VMSA family members, including msa-1. Moreover, all but one of the Australian breakthrough isolate MSA-2a/b proteins is markedly different from the vaccine strain from which immune escape occurred, consistent with their role in strain-specific protective immunity. The densest distribution of polymorphisms occurs in a hypervariable region (HVR) within the carboxy third of the molecule that is highly proline rich. Variation in length and content of the HVR is primarily attributable to differences in the order and number of degenerate nucleotide repeats encoding three motifs of unknown function.

Strain-Specific T-Cell Suppression and Protective Immunity in Patients with Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) frequently persists with an apparently ineffective antiviral T-cell response. We hypothesized that some patients may be exposed to multiple HCV subtypes and that strain-specific T cells could contribute to the viral dynamics in this setting. To test this hypothesis, CD4 T-cell responses to three genotype 1a-derived HCV antigens and HCV antibody serotype were examined in chronically HCV infected (genotypes 1a, 1b, 2, 3, and 4) and spontaneously HCV recovered subjects. Consistent with multiple HCV exposure, 63% of patients infected with genotypes 2 to 4 (genotypes 2-4) and 36% of those infected with genotype 1b displayed CD4 T-cell responses to 1a-derived HCV antigens, while 29% of genotype 2-4-infected patients showed serotype responses to genotype 1. Detection of 1a-specific T cells in patients without active 1a infection suggested prior self-limited 1a infection with T-cell-mediated protection from 1a but not from non-1a viruses. Remarkably, CD4 T-cell responses to 1a-derived HCV antigens were weakest in patients with homologous 1a infection and greater in non-1a-infected patients: proportions of patients responding were 19% (1a), 36% (1b), and 63% (2-4) (P = 0.0006). Increased 1a-specific CD4 T-cell responsiveness in non-1a-infected patients was not due to increased immunogenicity or cross-reactivity of non-1a viruses but directly related to sequence divergence. We conclude that the T-cell response to the circulating virus is either suppressed or not induced in a strain-specific manner in chronically HCV infected patients and that, despite their ability to clear one HCV strain, patients may be reinfected with a heterologous strain that can then persist. These findings provide new insights into host-virus interactions in HCV infection that have implications for vaccine development.

Parasite genetics and the immune host: recombination between antigenic types of Eimeria maxima as an entrée to the identification of protective antigens

The genomes of protozoan parasites encode thousands of gene products and identification of the subset that stimulates a protective immune response is a daunting task. Most screens for vaccine candidates identify molecules by capacity to induce immune responses rather than protection. This paper describes the core findings of a strategy developed with the coccidial parasite Eimeria maxima to rationally identify loci within its genome that encode immunoprotective antigens. Our strategy uses a novel combination of parasite genetics, DNA fingerprinting, drug-resistance and strain-specific immunity and centres on two strains of E. maxima that each induce a lethal strain-specific protective immune response in the host and show a differential response to anti-Eimeria chemotherapy. Through classical mating studies with these strains we have demonstrated that loci encoding molecules stimulating strain-specific protective immunity or resistance to the anti-coccidial drug robenidine segregate independently. Furthermore, passage of populations of recombinant parasites in the face of killing in the immune host was accompanied by the elimination of some polymorphic DNA markers defining the parent strain used to immunise the host. Consideration of the numbers of parasites recombinant for the two traits implicates very few antigen-encoding loci. Our data provide a potential strategy to identify putative antigen-encoding loci in other parasites.

Modification of otitis media in chinchillas rechallenged with nontypable Haemophilus influenzae and serological response to outer membrane antigens.

Otitis media was produced in chinchillas by right-sided intrabullar inoculation with nontypable Haemophilus influenzae, and susceptibility to reinfection was investigated. After resolution of initial right-sided infection, animals underwent ipsilateral or contralateral intrabullar rechallenge with the same strain. After ipsilateral rechallenge right ears were completely protected against reinfection; previously uninfected left ears were similarly protected on contralateral rechallenge. Previously infected ears remained fully susceptible to infection with a heterologous strain of nontypable H. influenzae. Using an enzyme-linked immunosorbent assay, we measured the serological response to outer membrane protein and lipopolysaccharide antigens during initial infection. A greater than or equal to 10-fold rise in titer of antibody to homologous outer membrane proteins was observed in all 11 animals tested. Most animals exhibited a minimal serological response to lipopolysaccharide. Thus experimental otitis media due to nontypable H. influenzae induces strain-specific protective immunity and a concomitant serological response to outer membrane proteins.

Do children with recurrent Haemophilus influenzae otitis media become infected with a new organism or reacquire the original strain?

Using the techniques of outer membrane protein gel analysis and biotyping, we characterized nontypable Haemophilus influenzae isolates from middle ear aspirates of 30 children with recurrent nontypable H, influenzae (NTHI) otitis media. Nine of the 13 children with early recurrence of NTHI otitis (less than 30-day intervals) had respective first and second isolates that were identical. In contrast, 14 of 18 children with late recurrences of NTHI otitis (greater than 30-day intervals) had respective first and second isolates that were different, whereas four children had late recurrences with organisms that appeared to be identical with their respective initial infecting strains. These results suggest that early recurrent NTHI otitis usually is a result of relapse with the initial infecting NTHI strain. In contrast, late recurrent disease is usually the result of infection with a new organism. However, the observation that four children had late recurrences with the original strains suggests that strain-specific protective immunity may not uniformly develop after recovery from NTHI otitis.

Strain Specific Protective Immunity against Trypanosoma cruzi

Swiss albino, C57BL/10J, and B10.A mice previously inoculated with an LD50 dose of T. cruzi Y strain were protected against subsequent challenge with the homologous strain, but had a parasitemia comparable to that of control mice when challenged with F strain trypomastigotes, although they were protected against mortality. In contrast, animals previously inoculated with an LD50 of T. cruzi F strain were protected both against a subsequent challenge with the homologous strain and the Y strain.