Reduced immunogenicity of Plasmodium falciparum gamete surface antigen (Pfs48/45) in mice after disruption of disulphide bonds – evaluating effect of interferon‐γ‐inducible lysosomal thiol reductase

Sexual stages of Plasmodium are critical for malaria transmission and stage‐specific antigens are important targets for development of malaria transmission‐blocking vaccines. Plasmodium falciparum gamete surface antigen (Pfs48/45) is important for male gamete fertility and is being pursued as a candidate vaccine antigen. Vaccine‐induced transmission‐blocking antibodies recognize reduction‐sensitive conformational epitopes in Pfs48/45. Processing and presentation of such disulphide‐bond‐constrained epitopes is critical for eliciting the desired immune responses. Mice lacking interferon‐γ‐inducible lysosomal thiol reductase (GILT), an enzyme that mediates reduction of S‐S bonds during antigen processing, were employed to investigate immunogenicity of Pfs48/45. It has been well established that the ability to reduce S‐S bonds in antigens guides effective T‐cell immune responses; however, involvement of GILT in the induction of subsequent B‐cell responses has not been explored. We hypothesized that the ability to reduce S‐S bonds in Pfs48/45 will impact the generation of T‐cell epitopes, and so influence helper T‐cell responses required for specific B‐cell responses. Non‐reduced and reduced and alkylated forms of Pfs48/45 were employed to evaluate immune responses in wild‐type and GILT knockout mice and studies revealed important differences in several immune response parameters, including differences in putative T‐cell epitope recognition, faster kinetics of waning of Pfs48/45‐specific IgG1 antibodies in knockout mice, differential patterns of interferon‐γ and interleukin‐4 secretions by splenocytes, and possible effects of GILT on induction of long‐lived plasma cells and memory B cells responsible for antigen‐recall responses. These studies emphasize the importance of antigen structural features that significantly influence the development of effective immune responses.     

Naturally acquired immune responses to Plasmodium falciparum sexual stage antigens Pfs48/45 and Pfs230 in an area of seasonal transmission

Acquisition of immunity to Plasmodium falciparum sexual stages is a key determinant for reducing human-mosquito transmission by preventing the fertilization and the development of the parasite in the mosquito midgut. Naturally acquired immunity against sexual stages may therefore form the basis for the development of transmission-blocking vaccines, but studies conducted to date offer little in the way of consistent findings. Here, we describe the acquisition of antigametocyte immune responses in malaria-exposed individuals in Burkina Faso. A total of 719 blood samples were collected in a series of three cross-sectional surveys at the start, peak, and end of the wet season. The seroprevalence of antibodies with specificity for the sexual stage antigens Pfs48/45 and Pfs230 was 2-fold lower (22 to 28%) than that for an asexual blood stage antigen glutamate-rich protein (GLURP) (65%) or for the preerythrocytic stage antigen circumsporozoite protein (CSP) (54%). The youngest children responded at frequencies similar to those for all four antigens but, in contrast with the immune responses to GLURP and CSP that increased with age independently of season and area of residence, there was no evidence for a clear age dependence of responses to Pfs48/45 and Pfs230. Anti-Pfs230 antibodies were most prevalent at the peak of the wet season (P < 0.001). Our findings suggest that naturally acquired immunity against Pfs48/45 and Pfs230 is a function of recent exposure rather than of cumulative exposure to gametocytes.     

Complement-mediated lysis of Plasmodium falciparum gametes by malaria-immune human sera is associated with antibodies to the gamete surface antigen Pfs230.

Antibodies to the sexual-stage surface antigens of Plasmodium falciparum, Pfs230 and Pfs48/45, can abolish the infectivity of gametes to mosquitoes; these antigens have been proposed as candidates for inclusion in a malaria transmission-blocking vaccine. One possible mechanism of antibody-mediated transmission blocking is complement-mediated gamete lysis. We have used a panel of human sera from geographically distinct regions where malaria is endemic to investigate whether this may be a mechanism of naturally acquired transmission-blocking immunity to P. falciparum. By immunoprecipitation, we have shown that antibody recognition of Pfs230 and Pfs48/45 is limited, despite universal exposure to P. falciparum gametocytes. In vitro complement-mediated lysis of P. falciparum gametes was positively associated with the presence of antibodies to Pfs230 but not with antibodies to the N-terminal region of the precursor molecule (Pfs260), which is shed from the gametocyte surface at the time of gametogenesis. Similarly, antibodies to two other gametocyte-specific proteins, Pfs48/45 and Pfg27/25, were not associated with gamete lysis. All sera which mediate gamete lysis contain immunoglobulin G1 (IgG1) and/or IgG3 antibodies to gamete surface proteins as determined by an enzyme-linked immunosorbent assay. These data suggest that Pfs230 is a major target of complement-fixing antibodies which may be important for antibody-mediated transmission-blocking immunity.     

Nasal immunization with a malaria transmission-blocking vaccine candidate, Pfs25, induces complete protective immunity in mice against field isolates of Plasmodium falciparum

Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.     

恶性疟原虫海南分离株FCC1/HN有性期特异抗原Pfs48/45基因的克隆与表达

采用基因工程技术,将编码恶性疟原虫有性期特异抗原Ｐｆｓ８／４５的基因克隆到真核表达质粒ｐｃＤ－ＮＡ３,并进行ＤＮＡ序列测定,再通过磷酸钙—ＤＮＡ共沉淀转化法将重组质粒ｐｃＤＮＡ３－ｐＦＳ４８／４５导入ＨｅＬａ细胞,建立稳定分泌Ｐｆｓ４８／４５蛋白的阳性克隆株。结果显示,我国海南ＦＣＣ１／ＨＮ株Ｐｆｓ４８／４５抗原基因序列与ＮＦ５４株者高度同源,提示该基因在不同虫株间高度保守,是研制疟疾疫苗的理想靶抗原;在ＨｅＬａ细胞中表达的Ｐｆｓ４８／４５蛋白分子量约为４６／４３．５ｋＤａ双联体蛋白,其表达量占细胞培养上清蛋白总量的１８．２７％。经ＷｅｓｔｅｒｎＢｌｏｔ分析显示,表在蛋白能被配子体免疫鼠血清特异性识别,提示表达的重组蛋白Ｐｆｓ４８／４５具有免疫活性。真核表达系统ｐｃＤＮＡ３／Ｐｆｓ４８／４５／ＨｅＬａ的建立为进一步研究重组Ｐｆｓ４８／４５抗原的免疫原性和保护性奠定基础。     

Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification

Two quantitative nucleic acid sequence-based amplification assays (QT-NASBA) based on Pfs16 and Pfs25, have been developed to quantify sexual stage commitment and mature gametocytes of Plasmodium falciparum. Pfs16 mRNA is expressed in all sexual forms including sexually committed ring stages while expression of Pfs25 mRNA is restricted to late stage gametocytes. Both assays showed a sensitivity of one sexual stage parasite/microl of blood. Blood samples from experimentally infected non-immune human volunteers were tested for Plasmodium falciparum by standard microscopy, a previously developed asexual 18S rRNA QT-NASBA, Pfs16 and Pfs25 mRNA QT-NASBA. Pfs16 QT-NASBA was positive in 9 out of 10 volunteers within 48 h after first detection of 18S rRNA, mostly before or at the day of positive microscopy. In contrast, the Pfs25 mRNA QT-NASBA was negative during the 28 days of follow-up, but consistently positive in gametocyte samples from naturally infected Kenyan patients. These data suggest that sexual stage commitment can occur early in the blood-stage infection without successful maturation into infectious gametocytes. In conclusion, Pfs16 and Pfs25 QT-NASBA assays in combination with a previously developed asexual stage QT-NASBA allow for the separate quantification of all developmental stages present in the circulation. The application of sexual stage QT-NASBA assays may contribute to a better understanding of the biology and epidemiology of malaria transmission.     

Polymorphism in the gene encoding the Pfs48/45 antigen of Plasmodium falciparum. XI. Asembo Bay Cohort Project.

We have investigated the genetic diversity of the gene encoding the transmission-blocking vaccine antigen Pfs48/45 of Plasmodium falciparum parasites from western Kenya and compared it with parasite populations from Thailand, India, and Venezuela. We report 44 complete new sequences. Overall, the antigen is less polymorphic as compared with other pre-erythrocytic and blood stage antigens. Contrary to other P. falciparum antigens, the number of synonymous substitutions per synonymous site exceeds the number of non-synonymous substitutions per non-synonymous site. We have found that the Pfs48/45 gene of Kenyan parasites is more polymorphic than parasites from other geographic origins. Our analysis reveals that positive natural selection is involved in the maintenance of the observed polymorphism. No evidence of intragenic recombination was found. F(st) values reveal high levels of gene flow between India and Thailand, however, there are strong constraints in gene flow among Kenyan, Southeast Asian, and Venezuelan parasites. No alleles could be linked to a specific geographic region. The results of this study suggest that this gametocyte antigen, like other asexual blood stage antigens, is under selection pressure.     

Phagocytosis does not play a major role in naturally acquired transmission-blocking immunity to Plasmodium falciparum malaria

Phagocytosis of Plasmodium falciparum sexual stages in vitro and within the mosquito midgut was assayed in order to assess its role in transmission-blocking immunity to malaria. Both monocytes/macrophages (MM) and polymorphonuclear neutrophils (PMN) phagocytosed malarial gametes in vitro, but levels of phagocytosis were low. Intraerythrocytic gametocytes were not susceptible to phagocytosis. In vitro phagocytosis was positively correlated with levels of antibodies against the gamete surface proteins Pfs230 and Pfs48/45. Immunoglobulin G (IgG) subclass analysis revealed that phagocytosis was correlated with levels of antigamete IgG1. In vivo membrane-feeding experiments were performed in the presence of both pooled and individual malaria immune sera. The phagocytic process proceeded less efficiently in vivo than in vitro, which may be related to the lower ambient temperature (26 degrees C, compared with 37 degrees C). Finally, although we found a correlation between the ability of a serum to promote phagocytosis in vitro and the presence of antibodies against transmission-blocking target antigens, we were unable to demonstrate a role for MM- or PMN-mediated phagocytosis in reduction of infectivity of the malarial parasite to mosquitoes.     

Synthetic antigens representing the antigenic variation of human hepatitis C virus

Immune responses against hepatitis C virus (HCV) have been studied by numerous groups. However, details concerning the production of antibodies to antigenically variable epitopes remain to be elucidated. Since the sequences of the variable regions of several HCV proteins are different among the virus strains infecting patients, we decided to design peptide combinations that represent the theoretical maximum antigenic variation of each epitope to be used as capture antigens. We prepared six peptide mixtures (hypervariable epitope constructs; HECs) representing six different epitopes from structural and non-structural proteins of HCV from genotypes 1-6. Plasma from 300 HCV patients was tested to determine if their antibodies recognize the synthetic constructs. All the patients were chronically infected with diverse HCV genotypes and did not receive antiviral treatment. Antibodies to one or more of the HECs were detected in all of the HCV-infected individuals. Immunogenicity of the HCV HECs was also evaluated in outbred and inbred mice. Strong HEC-specific antibodies were produced, and cellular responses were also induced that were Th-1 rather than Th-2. Our results show that HCV HECs are both antigens that can be used to detect the broad cross-reactivity of antibodies from HCV-infected patients, and strong immunogens that can induce antigen-specific humoral and cellular immune responses in mice.     

Recombinant vaccinia viruses for the characterization of Plasmodium falciparum-specific cytotoxic T lymphocytes: recognition of processed antigen despite limited re-stimulation efficacy

Cytotoxic T lymphocytes (CTL) have been implicated in immunity to Plasmodium falciparum infection and disease. We have previously described the use of peptides to define malaria-specific CTL epitopes. To determine whether these peptide epitopes are processed intracellularly from the whole antigen we have developed recombinant vaccinia viruses (rVV) expressing three malaria antigens: thrombospondin-related adhesive protein (TRAP), Pfs16 and the C- terminal half of liver-stage antigen (LSA)-1. Target cells infected with recombinant viruses were lysed by malaria-specific CTL from semi- immune African donors. We also tested the ability of cells infected with these recombinant vaccinia viruses to re-stimulate malaria- specific CTL in peripheral blood lymphocytes from malaria immune adults. Two other pox virus recombinants, NYVAC, an attenuated vaccinia virus, and ALVAC, a canarypox virus, both expressing malaria antigens were also evaluated for their ability to stimulate malaria-specific CTL in contrast to peptide, none of these viruses successfully re- stimulated CTL from the peripheral blood lymphocytes of semi-immune donors. The ability of human CTL from naturally exposed individuals to recognize processed antigen supports the relevance of these cells in protective immunity to malaria.      

Evidence of non-neutral polymorphism in Plasmodium falciparum gamete surface protein genes Pfs47 and Pfs48/45

Targeted disruption of particular members of the Plasmodium 6-cys protein gene family, including Ps47, Ps48/45 and Ps230, is known to dramatically affect parasite fertility. Because loci critical to fertility in many eukaryote species have been shown to be under strong positive selection, we examined sequence variation in four members of the 6-cys protein gene family in Plasmodium falciparum (Pfs36, Pfs38, Pfs47 and Pfs48/45) to determine whether genetic variation in these loci may be of functional significance. Sequence polymorphism among 11 laboratory isolates of P. falciparum was compared with divergence from the respective orthologues in the closely related species P. reichenowi, showing an almost significant skew towards within-species non-synonymous polymorphism in Pfs47 and Pfs48/45 (by the McDonald-Kreitman test) but clearly non-significant results for Pfs36 and Pfs38. A preliminary analysis of Pfs47 sequence polymorphism in field isolates of P. falciparum showed exceptionally high fixation indices (F(ST)) among geographically distinct populations, similar to results seen previously for Pfs48/45. Therefore, both Pfs47 and Pfs48/45 were further analysed by sequencing polymorphic parts of the genes from a Tanzanian population sample of oocysts (a means of analysing diploid genotypes). Both genes displayed higher inbreeding coefficients (F(IS)) compared with the average of 11 unlinked microsatellite loci. These results suggest that allelic variation in these two genes may be functionally significant in influencing mating interactions, a hypothesis that could be tested by fertilization experiments with targeted allelic replacement.     

Immunogenicity of Plasmodium falciparum sexual stage antigens: implications for the design of a transmission blocking vaccine.

Immunogenicity of sexual stage antigens and boosting of transmission blocking antibodies following a natural infection are two critical factors in the design of an effective, subunit vaccine to block the transmission of malaria from man to mosquito. Immunogenicity and boosting are both T cell-dependent. Antigens, such as the 230-kDa, the 48/45-kDa, and the 40/10-kDa, expressed early in the extracellular forms of the sexual stage of Plasmodium falciparum, have limited immunogenicity in humans and in mice. In contrast, Pfs25, expressed predominantly in zygotes and ookinetes, has widespread immunogenicity in mice. Pfs25 expressed by a recombinant vaccinia virus (vSIDK) is also widely immunogenic in mice, and induces transmission blocking antibodies following multiple inoculations with vSIDK. The implications of these immunogenicity data are discussed relative to the design of an effective transmission blocking vaccine.     

Specificities of antibodies that inhibit merozoite dispersal from malaria-infected erythrocytes

When malaria schizont-infected erythrocytes are cultured with immune serum, antibodies prevent dispersal of merozoites, resulting in the formation of immune clusters of merozoites (ICM) and inhibition of parasite growth. Antigens recognized by these antibodies were identified by probing two dimensional immunoblots of Plasmodium falciparum antigens with antibodies dissociated from immune complexes present at the surface of merozoites in ICM. Total immune serum recognized 88 of the 135 protein spots detected by colloidal gold staining, but antibodies dissociated from immune complexes recognized only 15 protein spots attributable to no more than eight distinct antigens. Antigens recognized by antibodies that inhibit merozoite dispersal include the precursor to the major merozoite surface antigens (gp195), a 126-kDa serine-repeat antigen (SERA), the 130-kDa protein that appears to bind to glycophorin (GBP130), and the approx. 45-kDa merozoite surface antigen. One other antigen (230/215-kDa doublet) was identified by using antibodies affinity purified from recombinant expression proteins. The identities of the other three antigens (150 kDa, 127 kDa and less than 30 kDa) were not determined. This approach provides a strategy for identifying epitopes accessible at the merozoite surface which may be important components of a multivalent vaccine against blood stages of P. falciparum.     

Human antibody responses to Schistosoma mansoni: the influence of epitopes shared between different life-cycle stages on the response to the schistosomulum

Sera from 120 Kenyan schoolchildren who were infected with S. mansoni were individually examined, using an enzyme-linked immunoabsorbent assay (ELISA), for the presence of IgG and IgM antibodies reactive with antigens derived from adult worms, the outer membrane of the schistosomulum or from the parasite egg. In addition, antibodies against more purified egg antigens, an egg stage-specific glycoprotein preparation and a polysaccharide egg antigen known to share epitopes with the schistosomular surface were measured in ELISA, as were antibodies reactive with trichloroacetic acid-soluble and periodate-insensitive antigens derived from the outer membrane of schistosomulum and antigens shed when schistosomula were cultured in vitro. IgG subclass responses to the unfractionated egg antigen were also measured. The results from each of these assays were compared with the results of each other assay and with the number of parasite eggs excreted by each child, using Spearmans rank correlations. These comparisons revealed a number of statistically significant positive correlations. IgG4 anti-egg antibodies correlated better with intensity of infection than did other IgG subclasses. Total IgG responses against polysaccharide antigens did not correlate with intensity of infection as well as IgG responses against other antigens; epitopes shared between the schistosomulum surface and the adult worm were different to those shared with the parasite egg; and, there was antigen-directed restriction of IgG subclass responses to some egg and adult worm antigens which carried these shared epitopes. It is argued that this might have a qualitative effect on the nature of the antibodies directed against the schistosomulum by infected individuals and therefore have important consequences for the outcome of a subsequent exposure to infection with the same parasite.     

Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi’s sarcoma

 Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi’s sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8. Received: 3 August 1996 / Accepted: 28 November 1996     

Further characterization of interactions between gamete surface antigens of Plasmodium falciparum.

Target antigens of malaria transmission blocking immunity include a complex of 3 gamete surface proteins of 230-kDa and 48/45-kDa glycoproteins. Previous studies have shown that epitopes recognized by blocking antibodies are conformational (reduction sensitive) in nature. Studies were conducted to characterize the interactions between the target antigens and role of disulfide groups in the formation of the complex. Treatment of detergent extracts of gametes with chaotropic agents and extremes of pH resulted in dissociation of the complex. The interaction between the 3 proteins was also perturbed when the extract was incubated in the presence of antibodies against the 230-kDa protein but not against the 48/45-kDa doublet. Chemical modifications of disulfide and sulfhydryl groups in the target antigens, otherwise inaccessible either in the total extract or after phase separation in Triton X-114, required prior denaturation of antigens.     

Pfs47, paralog of the male fertility factor Pfs48/45, is a female specific surface protein in Plasmodium falciparum

The genome of Plasmodium falciparum contains a small gene family that expresses proteins characterized by the presence of 6-cysteine domains. Most of these proteins are expressed on the surface of the parasite and some are known to play a role in cell-cell interactions. Two members of this family, Pfs48/45 and Pfs230, form a complex localized on the surface of gametes and are recognized as important targets for transmission-blocking vaccines. In this study we report the analysis of an additional member of this family, Pfs47 the closest paralog of Pfs48/45. We demonstrate that Pfs47 is expressed only in female gametocytes and is located on the surface of female gametes following emergence from red blood cells. In contrast to the critical function of P48/45 for male fertility, Pfs47 does not appear crucial for female fertility. Parasites lacking Pfs47 through targeted gene disruption, produce normal numbers of oocysts when included in the blood meal of the mosquito vector. In addition, three monoclonal antibodies against Pfs47 were unable to inhibit oocyst development when present in a blood meal containing wild type parasites. These results show redundancy in protein function for Pfs47 and reduce the support for candidacy of Pfs47 as a transmission-blocking vaccine target.     

Association between HLA type and antibody response to malaria sporozoite and gametocyte epitopes is not evident in immune Papua New Guineans.

HLA-A,B,C and DR types were determined for 46 adults living in the Madang area of Papua New Guinea. Sera from these individuals were tested by ELISA for antibodies against: (i) sonicated schizont extract of Plasmodium falciparum; (ii) circumsporozoite repeat regions of P. falciparum and P. vivax; and (iii) epitopes on the 230 and 48/45 kD gametocyte antigens of P. falciparum. All sera were from highly immune individuals and reacted strongly to the schizont antigen. The proportions responding to circumsporozoite repeat regions were 60.7% and 23.9% for P. falciparum and P. vivax, respectively. Between 32.6 and 47.8% of adults responded to each gametocyte epitope as assessed by inhibition of monoclonal antibodies. The limited number of alleles present at each HLA locus which is characteristic of coastal Papua New Guinea was observed. Five HLA-DR alleles were detected, of which only three (HLA-DR2, 4 and w5) were present at frequencies over 0.12. All individuals possessed at least one DR2,4 or w5 allele, and 96% of individuals possessed DR2, or 4 or both. There was no evidence for association between HLA type and antibody response to circumsporozoite repeat regions or the gametocyte epitopes. Homozygotes for DR2 and 4 were able to respond to each antigen. These results imply that either there is no HLA restriction of the response to these antigens or that each DR type is responding to a different variant of the T-epitope. Even in the latter case the results are encouraging for the prospects of inclusion of an HLA-restricted T-epitope in a malaria vaccine for Papua New Guinea since a limited number of versions would be required to cover a population with an HLA profile similar to that in Madang.     

Saccharomyces cerevisiae-Secreted Fusion Proteins Pfs25 and Pfs28 Elicit Potent Plasmodium falciparum Transmission-Blocking Antibodies in Mice

Transmission-blocking vaccines based on sexual-stage surface antigens of Plasmodium falciparum may assist in the control of this lethal form of human malaria. Two vaccine candidates, Pfs25 and Pfs28, were produced as single recombinant fusion proteins. The 39-kDa chimeric proteins, having a C-terminal His₆ tag, were secreted by Saccharomyces cerevisiae, using the prepro-α-factor leader sequence. Pfs25-28 fusion proteins were significantly more potent than either Pfs25 or Pfs28 alone in eliciting antibodies in mice that blocked oocyst development in Anopheles freeborni mosquitoes: complete inhibition of oocyst development in the mosquito midgut was achieved with fewer vaccinations, at a lower dose, and for a longer duration than with either Pfs25 or Pfs28 alone. Increased antigen-specific immunoglobulin G titers and highly significant lymphoproliferative stimulation by Pfs28-containing antigens suggest the presence of an immunodominant helper T-cell epitope in the Pfs28 portion of the fusion proteins. This epitope may be responsible for the enhanced humoral response to both Pfs25 and Pfs28 antigens. Protein production of the fusion protein was improved 12-fold by converting Pfs28 codons to yeast-preferred codons (TBV28), using a modified ADH₂ promoter and incorporating a (Glu-Ala)₂ repeat after the Kex2 cleavage site.The old tools of control, namely, vector control and chemotherapy, have been insufficient to reverse the increase in disease and death from malaria. They have proven to be neither cost-effective to deploy nor easy to sustain. Malaria vaccines hold the hope of an affordable, sustainable intervention that can be added to our armamentarium to fight this ancient scourge. Transmission-blocking vaccines that prevent the spread of vaccine-escape mutants and diminish the infectivity of the parasite to the mosquito are a potentially powerful component of malaria vaccines.Transmission of Plasmodium falciparum occurs only if parasites in infected blood ingested by a female Anopheles mosquito undergo sexual and sporogonic development within the mosquito midgut. By using an ex vivo membrane-feeding assay and monoclonal antibodies, several sexual-stage surface antigens have been shown to be targets of transmission-blocking immunity (3, 7, 8, 15, 18). By preventing the development of the parasite within the mosquito midgut, antibodies to these target antigens block transmission of P. falciparum to the mosquito vector. Pfs25, a 25-kDa surface antigen of zygotes and ookinetes (10), and Pfs28, a 28-kDa surface antigen of late ookinetes (4), are two of the lead vaccine candidates. Both comprise four tandem epidermal growth factor (EGF)-like domains presumably anchored to the parasite surface by glycosylphosphatidylinositol, and both, produced as recombinant proteins in yeast, have been shown to induce transmission-blocking antibodies in experimental laboratory animals (1, 11, 12).The mechanisms by which polyclonal sera and monoclonal antibodies to Pfs25 block transmission appear to reside in inhibiting the transformation of zygotes to ookinetes and the ability of ookinetes to infect mosquitoes, respectively (12). Polyclonal sera to Pgs28, the avian malaria parasite (P. gallinaceum) analog of Pfs28, appear to block transmission by inhibiting the transformation of zygotes to ookinetes in vitro and by suppressing the development of ookinetes to oocysts in vivo (5). Because antibodies to these two antigens appear to block infectivity by attacking different proteins on the same developmental stages, the question arose as to whether Pfs25 and Pfs28 will act synergistically to block parasite infectivity to mosquitoes. Indeed, mixing Pfs25 and Pfs28 mouse antibodies demonstrated a more potent transmission-blocking effect (4); however, combining yeast-produced Pfs25 and Pfs28 in a single vaccine did not produce a synergistic transmission-blocking effect in mice (8a). We report here that a fusion of Pfs25 and Pfs28 as a single chimeric recombinant protein is a more potent transmission-blocking vaccine than either antigen alone but perhaps not by antibodies to Pfs25 and Pfs28 acting synergistically.     

Induction of cross-neutralizing antibodies by sequential immunization with heterologous papillomavirus L1VLPs and its implications for HPV prophylactic vaccines

Sequential immunization with antigens from different strains of HIV-1, influenza viruses or dengue viruses induced cross-neutralizing antibodies and enhanced the antibody responses against previous antigens. The characteristics of neutralizing antibodies induced by sequential immunization with different types of human papillomavirus (HPV) L1 virus-like particles (L1VLPs) are unclear. In this study, mice were primed with one or two types (HPV-16 or HPV16/18) of L1VLPs, then boosted sequentially with HPV6/18/45/11/31/58 or HPV6/45/11/31/58 L1VLPs, and sera were analyzed with HPV pseudovirus-based neutralization assay. The results showed that neutralizing activities against earlier immunized vaccine types were enhanced gradually by subsequent immunizations, and low levels of neutralizing activities against nonvaccine types (HPV33/35/52/59/68) were also observed. After absorbing the immune sera with vaccine-type (HPV16/18/45) L1VLPs, neutralizing activities against tested priming and boosting types (HPV16/18/58) decreased significantly, and that against nonvaccine type (HPV-33) was also partially eliminated. Moreover, neutralizing activities against vaccine types (HPV16/58) were significantly reduced after absorbing with nonvaccine-type VLPs (HPV33/52). These data suggest that cross-neutralizing epitopes exist among different HPV L1VLPs. The cross-neutralizing activities against nonvaccine types and the enhanced neutralizing activities against earlier immunized vaccine types may result from sequential boosting with these cross-neutralizing epitopes. These observations support early vaccination with more types of L1VLPs derived from HPVs that cause a serious threat to the population.