Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.

https://iai.asm.org/content/iai/77/3/1165.full.pdf

Immunoglobulin G Subclass-Specific Responses against Plasmodium falciparum Merozoite Antigens Are Associated with Control of Parasitemia and Protection from Symptomatic Illness

Macaques have served as models for more than 70 human infectious diseases of diverse etiologies, including a multitude of agents-bacteria, viruses, fungi, parasites, prions. The remarkable diversity of human infectious diseases that have been modeled in the macaque includes global, childhood, and tropical diseases as well as newly emergent, sexually transmitted, oncogenic, degenerative neurologic, potential bioterrorism, and miscellaneous other diseases. Historically, macaques played a major role in establishing the etiology of yellow fever, polio, and prion diseases. With rare exceptions (Chagas disease, bartonellosis), all of the infectious diseases in this review are of Old World origin. Perhaps most surprising is the large number of tropical (16), newly emergent (7), and bioterrorism diseases (9) that have been modeled in macaques. Many of these human diseases (e.g., AIDS, hepatitis E, bartonellosis) are a consequence of zoonotic infection. However, infectious agents of certain diseases, including measles and tuberculosis, can sometimes go both ways, and thus several human pathogens are threats to nonhuman primates including macaques. Through experimental studies in macaques, researchers have gained insight into pathogenic mechanisms and novel treatment and vaccine approaches for many human infectious diseases, most notably acquired immunodeficiency syndrome (AIDS), which is caused by infection with human immunodeficiency virus (HIV). Other infectious agents for which macaques have been a uniquely valuable resource for biomedical research, and particularly vaccinology, include influenza virus, paramyxoviruses, flaviviruses, arenaviruses, hepatitis E virus, papillomavirus, smallpox virus, Mycobacteria, Bacillus anthracis, Helicobacter pylori, Yersinia pestis, and Plasmodium species. This review summarizes the extensive past and present research on macaque models of human infectious disease.

/pdf/macaque-models-of-human-infectious-disease-224f178hv5.pdf

Macaque models of human infectious disease.

More attention is being focused on malaria today than any time since the world's last efforts to achieve eradication over 40 years ago. The global community is now discussing strategies aimed at dramatically reducing malarial disease burden and the eventual eradication of all types of malaria, everywhere. As a consequence, Plasmodium vivax, which has long been neglected and mistakenly considered inconsequential, is now entering into the strategic debates taking place on malaria epidemiology and control, drug resistance, pathogenesis and vaccines. Thus, contrary to the past, the malaria research community is becoming more aware and concerned about the widespread spectrum of illness and death caused by up to a couple of hundred million cases of vivax malaria each year. This review brings these issues to light and provides an overview of P. vivax vaccine development, then and now. Progress had been slow, given inherent research challenges and minimal support in the past, but prospects are looking better for making headway in the next few years. P. vivax, known to invade the youngest red blood cells, the reticulocytes, presents a strong challenge towards developing a reliable long-term culture system to facilitate needed research. The P. vivax genome was published recently, and vivax researchers now need to coordinate efforts to discover new vaccine candidates, establish new vaccine approaches, capitalize on non-human primate models for testing, and investigate the unique biological features of P. vivax, including the elusive P. vivax hypnozoites. Comparative studies on both P. falciparum and P. vivax in many areas of research will be essential to eradicate malaria. And to this end, the education and training of future generations of dedicated "malariologists" to advance our knowledge, understanding and the development of new interventions against each of the malaria species infecting humans also will be essential.

/pdf/plasmodium-vivax-who-cares-20dagxl396.pdf

Plasmodium vivax: who cares?

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

Population studies show that individuals acquire immunity to Plasmodium vivax more quickly than Plasmodium falciparum irrespective of overall transmission intensity, resulting in the peak burden of P. vivax malaria in younger age groups. Similarly, actively induced P. vivax infections in malaria therapy patients resulted in faster and generally more strain-transcending acquisition of immunity than P. falciparum infections. The mechanisms behind the more rapid acquisition of immunity to P. vivax are poorly understood. Natural acquired immune responses to P. vivax target both pre-erythrocytic and blood-stage antigens and include humoral and cellular components. To date, only a few studies have investigated the association of these immune responses with protection, with most studies focussing on a few merozoite antigens (such as the Pv Duffy binding protein (PvDBP), the Pv reticulocyte binding proteins (PvRBPs), or the Pv merozoite surface proteins (PvMSP1, 3 & 9)) or the circumsporozoite protein (PvCSP). Naturally acquired transmission-blocking (TB) immunity (TBI) was also found in several populations. Although limited, these data support the premise that developing a multi-stage P. vivax vaccine may be feasible and is worth pursuing.

Natural acquisition of immunity to Plasmodium vivax: epidemiological observations and potential targets.

Progress towards a vaccine against Plasmodium falciparum malaria is advancing rapidly, with several candidate antigens being tested for safety and efficacy in humans (4); comparatively, however, the development of a vaccine against P. vivax malaria has lagged behind. Unlike P. falciparum, where sporozoite challenge studies using the chloroquine-sensitive 3D7 strain are available, the relapsing nature of P. vivax hepatic stages and the lack of an in vitro culture system precludes any sporozoite challenge studies of P. vivax vaccine candidates in humans. Therefore, as more recombinant vaccine products become available, there will be an increasing need to compare and down-select vaccine candidate antigens in preclinical studies using animal models of P. vivax infection.

P. cynomolgi, which infects rhesus macaques in southeast Asia, is a closely related species to P. vivax; human transmission of P. cynomolgi has also been reported (25). The two parasites share a similar clinical course of infection (26), a reticulocyte-specific invasion (17), the presence of Schuffner's dots on infected erythrocytes (2), and a dormant liver hypnozoite stage that is responsible for a relapsing blood stage infection (23). P. cynomolgi and P. vivax have similar genomic GC content, and rRNA analysis confirms their close taxonomic relatedness (43). High homology of prime vaccine candidates such as the apical membrane antigen 1, 97% (13); circumsporozoite protein, 90% (16); erythrocyte binding protein, 76% (32); and the 42-kDa fragment of the merozoite surface protein 1 (MSP-1), 75% (31); have been reported. Although it is believed that the P. cynomolgi-rhesus model can serve as a model for testing P. vivax antigens, there is only one previous report where this model was used to test the efficacy of a recombinant P. vivax vaccine (22).

The merozoite surface protein 1 (MSP-1), found on the surface of Plasmodium merozoites, has been a prime vaccine candidate for many years (10). Following its synthesis as a 200-kDa precursor, the MSP-1 molecule undergoes step-wise proteolytic processing resulting in a glycosyl-phosphatidylinositol-anchored 42-kDa protein (MSP-142) on the surface of free merozoites (7). This 42-kDa intermediate undergoes secondary processing at the time of invasion, releasing a 33-kDa soluble polypeptide (MSP-133) and leaving behind on the invading merozoite a glycosyl-phosphatidylinositol-anchored 19-kDa form (MSP-119) (7). Depending on the species, MSP-119 contains 10 or 11 cysteine residues that form five disulfide bonds. Immunization with recombinant MSP-142 and MSP-119 raises antibodies that inhibit invasion of merozoites in vitro (9) and protects the immunized animals against live parasite challenge (29).

We have previously reported a process for the production of correctly folded human vaccine-grade P. vivax MSP-142 protein in the bacterial expression host Escherichia coli Origami(DE3) (14). This bacterial strain has mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which greatly enhances the disulfide bond formation of recombinant proteins expressed in its cytoplasm, hence resulting in a soluble product (6). However, the transposon-mediated genetic modifications of E. coli carried both the tetracycline and the kanamycin resistance genes as selectable markers and therefore made the cells incompatible with the production of human use vaccines that relied on plasmids carrying the Tetr or the Kanr genes. The use of a compatible expression host E. coli BL21(DE3) resulted in the production of an MSP-142 protein that was insoluble and located in the inclusion body fraction of the cells. In this report we have shown that soluble MSP-142 protein can be obtained from the BL21(DE3) cells by in vitro refolding of the inclusion body-derived protein under controlled redox conditions. Both the soluble and refolded P. vivax MSP-142 products appeared to be structurally similar based on biophysical analysis and reactivity with conformational epitope-specific monoclonal antibodies.

It was our goal, therefore, to seek evidence of the immunological equivalence of these proteins in a parasite challenge model. Towards that end we report here the results of a vaccination study comparing the immunogenicity and efficacy of the soluble and refolded P. vivax MSP-142 products in the P. cynomolgi-rhesus model. We have also produced equivalent homologous constructs of P. cynomolgi MSP-142 protein both in their soluble and refolded forms, and these two proteins were also used as positive control immunogens in this study.

Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys.

Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine. P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-I (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-1 42 ) is present on the surface of merozoiles and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-1 42 ) and P. cynomologi B (PcMSP-1 42 ). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-1 42 proteins were homogenous in nature. The soluble and refolded MSP-1 42 antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP- 1 42 antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-1 42 protein. The immune sera from rhesus monkeys, immunized with soluble and refolded MSP-1 42 antigens of both parasites also showed high titered cross-reactive antibodies against MSP-1 42 conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P. vivax MSP-1 42 antigens were highly immunogenic and thus a viable candidate for vaccine studies.

http://nopr.niscair.res.in/bitstream/123456789/144/1/IJBB%2044%286%29%20%282007%29%20429-436.pdf

Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1.

Atul Narula

Papers

Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys.

Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1.