Transmission Blocking Vaccines Development Research Articles

A Novel Ex Vivo Assay to Evaluate Functional Effectiveness of Plasmodium vivax Transmission-Blocking Vaccine Using Pvs25 Transgenic Plasmodium berghei.

Plasmodium falciparum and Plasmodium vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs. Transgenic murine malaria parasites (Plasmodium berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA). We describe the development of transmission-competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen. The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax-infected patients in endemic areas for evaluation.

MRNA vaccines expressing malaria transmission-blocking antigens Pfs25 and Pfs230D1 induce a functional immune response

Malaria transmission-blocking vaccines (TBV) are designed to inhibit the sexual stage development of the parasite in the mosquito host and can play a significant role in achieving the goal of malaria elimination. Preclinical and clinical studies using protein–protein conjugates of leading TBV antigens Pfs25 and Pfs230 domain 1 (Pfs230D1) have demonstrated the feasibility of TBV. Nevertheless, other promising vaccine platforms for TBV remain underexplored. The recent success of mRNA vaccines revealed the potential of this technology for infectious diseases. We explored the mRNA platform for TBV development. mRNA constructs of Pfs25 and Pfs230D1 variously incorporating signal peptides (SP), GPI anchor, and Trans Membrane (TM) domain were assessed in vitro for antigen expression, and selected constructs were evaluated in mice. Only mRNA constructs with GPI anchor or TM domain that resulted in high cell surface expression of the antigens yielded strong immune responses in mice. These mRNA constructs generated higher transmission-reducing functional activity versus the corresponding alum-adjuvanted protein-protein conjugates used as comparators. Pfs25 mRNA with GPI anchor or TM maintained >99% transmission reducing activity through 126 days, the duration of the study, demonstrating the potential of mRNA platform for TBV.

Molecular Changes in Dengue Envelope Protein Domain III upon Interaction with Glycosaminoglycans.

Dengue fever is a rapidly emerging vector-borne viral disease with a growing global burden of approximately 390 million new infections per annum. The Dengue virus (DENV) is a flavivirus spread by female mosquitos of the aedes genus, but the mechanism of viral endocytosis is poorly understood at a molecular level, preventing the development of effective transmission blocking vaccines (TBVs). Recently, glycosaminoglycans (GAGs) have been identified as playing a role during initial viral attachment through interaction with the third domain of the viral envelope protein (EDIII). Here, we report a systematic study investigating the effect of a range of biologically relevant GAGs on the structure and oligomeric state of recombinantly generated EDIII. We provide novel in situ biophysical evidence that heparin and chondroitin sulphate C induce conformational changes in EDIII at the secondary structure level. Furthermore, we report the ability of chondroitin sulphate C to bind EDIII and induce higher-order dynamic molecular changes at the tertiary and quaternary structure levels which are dependent on pH, GAG species, and the GAG sulphation state. Lastly, we conducted ab initio modelling of Small Angle Neutron Scattering (SANS) data to visualise the induced oligomeric state of EDIII caused by interaction with chondroitin sulphate C, which may aid in TBV development.

Strong concordance between percent inhibition in oocyst and sporozoite intensities in a Plasmodium falciparum standard membrane-feeding assay

BackgroundEffective malaria transmission-blocking vaccines (TBVs) can support malaria eradication programmes, and the standard membrane-feeding assay (SMFA) has been used as a “gold standard” assay for TBV development. However, in SMFA, the inhibitory activity is commonly measured at oocyst stage of parasites, while it is the sporozoites which transmit malaria from a mosquito to a human. A handful of studies have shown that there is a positive correlation between oocyst and sporozoite intensities. However, no study has been completed to compare inhibition levels in oocyst and sporozoite intensities in the presence of transmission-blocking (TB) antibodies.ResultsPlasmodium falciparum NF54 gametocytes were fed to Anopheles stephensi mosquitoes with or without anti-Pfs25 or anti-Pfs48/45 TB antibodies in 15 independent assays. For each group, a portion of the mosquitoes was dissected for oocyst counts (day 8 after feed), and a portion of the remaining mosquitoes was dissected for sporozoite counts (day 16). This study covered a large range of oocyst and sporozoite intensities: 0.2 to 80.5 on average for oocysts, and 141 to 77,417 for sporozoites. The sporozoite data were well explained by a zero-inflated negative binomial model, regardless of the presence or absence of TB antibodies. Inhibition levels in both oocyst and sporozoite intensities were determined within the same groups in 9 independent assays. When the level of inhibition in sporozoite number (expressed as Log Mean Ratio, LMR; average number in a control group was divided by the one in a test group, then took a log of the ratio) was plotted against LMR in oocyst number, the best-fit slope of a linear regression was not different from 1 (the best estimate, 1.08; 95% confidence interval, 0.87 to 1.29). Furthermore, a Bland–Altman analysis showed a strong agreement between inhibitions in oocysts and in sporozoites.ConclusionsThe results indicate that percent inhibition in oocyst intensity of a test sample can be directly converted to % inhibition in sporozoite intensity in P. falciparum SMFA. Therefore, if sporozoite intensity determines transmission rate from mosquitoes to humans, the percent inhibition in oocyst intensity measured by SMFA can be used to estimate the TBV efficacy.

ELISA units, IgG subclass ratio and avidity determined functional activity of mouse anti-Pfs230 antibodies judged by a standard membrane-feeding assay with Plasmodium falciparum

The standard membrane-feeding assay (SMFA) is a functional assay that has been used to inform the development of transmission-blocking vaccines (TBV) against Plasmodium falciparum malaria. For Pfs230, a lead target antigen for TBV development, a few studies have tested either a single anti-Pfs230 polyclonal or monoclonal antibody (one antibody per study) at serial dilutions and showed a dose-dependent response. Further, there have been reports that the SMFA activity of anti-Pfs230 polyclonal and monoclonal antibodies were enhanced in the presence of complement. However, no analysis has been performed with multiple samples, and the impact of anti-Pfs230 antibody titers, IgG subclass profile and avidity were evaluated together in relation to transmission-reducing activity (TRA) by SMFA. In this report, a total of 39 unique anti-Pfs230 IgGs from five different mouse immunization studies were assessed for their ELISA units (EU), IgG2/IgG1 ratio and avidity by ELISA, and the functionality (% transmission-reducing activity, %TRA) by SMFA. The mice were immunized with Pfs230 alone, Pfs230 conjugated to CRM197, or a mixture of unconjugated Pfs230 and CRM197 proteins using Alhydrogel or Montanide ISA720 adjuvants. In all studies, the Pfs230 antigen was from the same source. There was a significant correlation between EU and %TRA (p < 0.0001 by a Spearman rank test) for the anti-Pfs230 IgGs. Notably, multiple linear regression analyses showed that both IgG2/IgG1 ratio and avidity significantly affected %TRA (p = 0.003 to p = 0.014, depending on the models) after adjusting for EU. The results suggest that in addition to antibody titers, IgG2/IgG1 ratio and avidity should each be evaluated to predict the biological activity of anti-Pfs230 antibodies for future vaccine development.

Safety and immunogenicity of a plant-produced Pfs25 virus-like particle as a transmission blocking vaccine against malaria: A Phase 1 dose-escalation study in healthy adults

Malaria continues to be one of the world’s most devastating infectious tropical diseases, and alternative strategies to prevent infection and disease spread are urgently needed. These strategies include the development of effective vaccines, such as malaria transmission blocking vaccines (TBV) directed against proteins found on the sexual stages of Plasmodium falciparum parasites present in the mosquito midgut. The Pfs25 protein, which is expressed on the surface of gametes, zygotes and ookinetes, has been a primary target for TBV development. One such vaccine strategy based on Pfs25 is a plant-produced malaria vaccine candidate engineered as a chimeric non-enveloped virus-like particle (VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein. This Pfs25 VLP-FhCMB vaccine candidate has been engineered and manufactured in Nicotiana benthamiana plants at pilot plant scale under current Good Manufacturing Practice guidelines. The safety, reactogenicity and immunogenicity of Pfs25 VLP-FhCMB was assessed in healthy adult volunteers. This Phase 1, dose escalation, first-in-human study was designed primarily to evaluate the safety of the purified plant-derived Pfs25 VLP combined with Alhydrogel® adjuvant. At the doses tested in this Phase 1 study, the vaccine was generally shown to be safe in healthy volunteers, with no incidence of vaccine-related serious adverse events and no evidence of any dose-limiting or dose-related toxicity, demonstrating that the plant-derived Pfs25 VLP-FhCMB vaccine had an acceptable safety and tolerability profile. In addition, although the vaccine did induce Pfs25-specific IgG in vaccinated patients in a dose dependent manner, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine adjuvant formulation.This study was registered at www.ClinicalTrials.gov under reference identifier NCT02013687.

Transmission-blocking activity is determined by transmission-reducing activity and number of control oocysts in Plasmodium falciparum standard membrane-feeding assay

Malaria transmission-blocking vaccines (TBVs) are potentially helpful tools for malaria eradication. The standard membrane-feeding assay (SMFA) is considered one of the “gold standard” assays for TBV development. However, lack of consensus in reporting results from SMFA has made it very challenging to compare results from different studies. Two main readouts, % inhibition in mean oocyst count per mosquito (TRA) and % inhibition in prevalence of infected mosquitoes (TBA), have been used widely.In this study, we statistically modeled the oocyst data in SMFA using data from 105 independent feeding experiments including 9804 mosquitoes. The model was validated using an independent data set that included 10,790 mosquitoes from 110 feeding studies. The model delineates a relationship between TRA, the mean oocyst count in the control mosquitoes (mo-contl), and TBA. While TRA was independent from mo-contl, TBA values changed depending on mo-contl. Regardless of monoclonal or polyclonal antibodies tested, there were strong concordances between observed TBA and predicted TBA based on the model using mo-contl and observed TRA. Simulations showed that SMFA with lower true control means had increased uncertainty in TRA estimates. The strong linkage between TBA, TRA and mo-contl inspired creation of a standardized TBA, a model-based TBA standardized to a target control mean, which allows comparison across multiple feeds regardless of mo-contl.This is the first study showing that the observed TBA can be reasonably predicted by mo-contl and the TRA of the test antibody using independent experimental data. This study indicates that TRA should be used to compare results from multiple feeds with different levels of mo-contl. If a measure of TBA is desired, it is better to report standardized TBA rather than observed TBA. These recommendations support rational comparisons of results from different studies, thus benefiting future TBV development.

Transmission blocking potency and immunogenicity of a plant-produced Pvs25-based subunit vaccine against Plasmodium vivax

Malaria transmission blocking (TB) vaccines (TBVs) directed against proteins expressed on the sexual stages of Plasmodium parasites are a potentially effective means to reduce transmission. Antibodies induced by TBVs block parasite development in the mosquito, and thus inhibit transmission to further human hosts. The ookinete surface protein P25 is a primary target for TBV development. Recently, transient expression in plants using hybrid viral vectors has demonstrated potential as a strategy for cost-effective and scalable production of recombinant vaccines. Using a plant virus-based expression system, we produced recombinant P25 protein of Plasmodium vivax (Pvs25) in Nicotiana benthamiana fused to a modified lichenase carrier protein. This candidate vaccine, Pvs25-FhCMB, was purified, characterized and evaluated for immunogenicity and efficacy using multiple adjuvants in a transgenic rodent model. An in vivo TB effect of up to a 65% reduction in intensity and 54% reduction in prevalence was observed using Abisco-100 adjuvant. The ability of this immunogen to induce a TB response was additionally combined with heterologous prime-boost vaccination with viral vectors expressing Pvs25. Significant blockade was observed when combining both platforms, achieving a 74% and 68% reduction in intensity and prevalence, respectively. This observation was confirmed by direct membrane feeding on field P. vivax samples, resulting in reductions in intensity/prevalence of 85.3% and 25.5%. These data demonstrate the potential of this vaccine candidate and support the feasibility of expressing Plasmodium antigens in a plant-based system for the production of TBVs, while demonstrating the potential advantages of combining multiple vaccine delivery systems to maximize efficacy.

A novel plant-produced Pfs25 fusion subunit vaccine induces long-lasting transmission blocking antibody responses

Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies. Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced transmission blocking antibodies that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system.