Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45.

Gamete surface protein P48/45 has been shown to be important for male gamete fertility and a strong candidate for the development of a malaria transmission-blocking vaccine (TBV). However, TBV development for Plasmodium vivax homolog Pvs48/45 has been slow because of a number of challenges: availability of conformationally suitable recombinant protein; the lack of an in vivo challenge model; and the inability to produce P. vivax gametocytes in culture to test transmission-blocking activity of antibodies. To support ongoing efforts to develop Pvs48/45 as a potential vaccine candidate, we initiated efforts to develop much needed reagents to move the field forward. We generated monoclonal antibodies (mAbs) directed against Pvs48/45 and characterized putative functional domains in Pvs48/45 using recombinant fragments corresponding to domains D1-D3 and their biological functionality through ex vivo direct membrane feeding assays (DMFAs) using P. vivax parasites from patients in a field setting in Brazil. While some mAbs partially blocked oocyst development in the DMFA, one mAb caused a significant enhancement of the infectivity of gametocytes in the mosquitoes. Individual mAbs exhibiting blocking and enhancing activities recognized non-overlapping epitopes in Pvs48/45. Further characterization of precise epitopes recognized by transmission-reducing and -enhancing antibodies will be crucial to design an effective immunogen with optimum transmission-reducing potential.

HIV-Related Stigma Research as a Priority at the National Institutes of Health.

The National Institutes of Health (NIH) recognizes that, despite HIV scientific advances, stigma and discrimination continue to be critical barriers to the uptake of evidence-based HIV interventions. Achieving the Ending the HIV Epidemic: A Plan for America (EHE) goals will require eliminating HIV-related stigma. NIH has a significant history of supporting HIV stigma research across its Institutes, Centers, and Offices (ICOs) as a research priority. This article provides an overview of NIH HIV stigma research efforts. Each ICO articulates how their mission shapes their interest in HIV stigma research and provides a summary of ICO-relevant scientific findings. Research gaps and/or future opportunities are identified throughout, with key research themes and approaches noted. Taken together, the collective actions on the part of the NIH, in tandem with a whole of government and whole of society approach, will contribute to achieving EHE's milestones.

RESUMEN: Los Institutos de Salud Nacional (NIH, siglas en inglés) reconocen que, a pesar de los avances en la prevención y el tratamiento, el estigma y la discriminación continúan siendo barreras críticas a la adopción de la prevención y el cuido basados en la evidencia. Las metas de Logrando el Fin de la Epidemia de VIH: Plan para América (EHE, siglas en inglés) requerirán la eliminación del estigma relacionado al VIH. Los NIH tienen una historia significativa apoyando la investigación del estigma relacionado al VIH a través de sus Institutos, Centros, y Oficinas (ICOs, siglas en inglés). Esta investigación es una prioridad fundamental y entrelazada para los ICOs. En este artículo, los autores de los NIH proveen una reseña sobre la investigación del estigma relacionado al VIH a través de los ICOs selectos. Cada ICO articula como su misión y prioridad dan forma a su interés en la investigación del estigma al VIH y provee una breve reseña de los hallazgos científicos pertinentes al ICO. Lagunas en la investigación relacionada a la misión, prioridades, y/o áreas de investigación futuras se identifican a través del artículo. También se apuntan en el resumen los temas de investigación claves y sus estrategias. En conjunto, las acciones colectivas de parte de los NIH, junto a la estrategia necesaria de parte del gobierno en su totalidad y de la sociedad en su totalidad, contribuirán al logro de las metas del EHE.

Impact of the Charge Ratio on the In Vivo Immunogenicity of Lipoplexes.

PURPOSE: The present study investigated the immunogenic potential of different cationic liposome formulations with a DNA plasmid encoding Pfs25, a malaria transmission-blocking vaccine candidate. METHODS: Pfs25 plasmid DNA was complexed with cationic liposomes to produce lipoplexes at different charge ratios of the cationic lipid head group to the nucleotide phosphate (N:P). The formation of lipoplexes was visualized by Cryogenic-TEM. Confocal microscopy of lipoplexes formed with GFP encoding plasmid DNA, and flow cytometry was used to determine their in vitro transfection capability. Two different lipoplex formulations using plasmid DNA encoding Pfs25 were evaluated for in vivo immunogenicity after intramuscular administration in Balb/c mice. Immune sera were analyzed by ELISA. RESULTS: The results demonstrated that the cationic liposome-mediated DNA immunization with an N:P charge ratio of 1:3 (anionic lipoplexes) is more effective than the use of naked plasmid DNA alone. No antibody response was observed when lipoplexes with a higher N:P charge ratio of 10:3 (cationic lipoplexes) were used. Trehalose was added to some lipoplex formulations as a cryoprotectant and adjuvant, but it did not yield any further improvement of immunogenicity in vivo. CONCLUSIONS: The results suggest that Pfs25 plasmid DNA delivered as lipoplexes at a charge ratio of 1:3 elicited strong immunogenicity in mice and may be improved further to match the immune responses of DNA vaccines administered by in vivo electroporation.

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.

Potent Functional Immunogenicity of Plasmodium falciparum Transmission-Blocking Antigen (Pfs25) Delivered with Nanoemulsion and Porous Polymeric Nanoparticles.

PURPOSE: To evaluate functional immunogenicity of CHrPfs25. a malaria transmission blocking vaccine antigen, using nanoemulsion and porous polymeric PLGA nanoparticles. METHODS: CHrPfs25 was formulated with nanoemulsions (NE) and poly(D,L-lactide-co-glycolide) nanoparticles (PLGA-NP) and evaluated via IM route in mice. Transmission blocking efficacy of antibodies was evaluated by standard mosquito membrane feeding assay using purified IgG from immune sera. Physicochemical properties and stability of various formulations were evaluated by measuring poly-dispersity index, particle size and zeta potential. RESULTS: Mice immunized with CHrPfs25 using alum via IP and IM routes induced comparable immune responses. The highest antibody response was obtained with CHrPfs25 formulated in 4% NE as compared to 8% NE and PLGA-NP. No further increases were observed by combining NE with MPL-A and chitosan. One hundred percent transmission blocking activity was demonstrated at 400 µg/ml of IgG for alum groups (both routes IP and IM), 4% NE and NE-MPL-A. Purified IgG from various adjuvant groups at lower doses (100 µg/mL) still exhibited >90% transmission blocking activity, while 52-81% blocking was seen at 50 µg/mL. CONCLUSION: Results suggest that CHrPfs25 delivered in various adjuvants/nanoparticles elicited strong functional immunogenicity in pre-clinical studies in mice. We are now continuing these studies to develop effective vaccine formulations for further evaluation of immune correlates of relative immunogenicity of CHrPfs25 in various adjuvants and clinical trials.

Immune mechanisms targeting malaria transmission: opportunities for vaccine development.

INTRODUCTION: Malaria continues to remain a major global health problem with nearly a quarter of a billion clinical cases and more than 600,000 deaths in 2022. There has been significant progress toward vaccine development, however, poor efficacy of approved vaccines requiring multiple immunizing doses emphasizes the need for continued efforts toward improved vaccines. Progress to date, nonetheless, has provided impetus for malaria elimination. AREAS COVERED: In this review we will focus on diverse immune mechanisms targeting gametocytes in the human host and gametocyte-mediated malaria transmission via the mosquito vector. EXPERT OPINION: To march toward the goal of malaria elimination it will be critical to target the process of malaria transmission by mosquitoes, mediated exclusively by the sexual stages, i.e. male, and female gametocytes, ingested from infected vertebrate host. Studies over several decades have established antigens in the parasite sexual stages developing in the mosquito midgut as attractive targets for the development of transmission blocking vaccines (TBVs). Immune clearance of gametocytes in the vertebrate host can synergize with TBVs and directly aid in maintaining effective transmission reducing immune potential.

Assets for integrating task-sharing strategies for hypertension within HIV clinics: Stakeholder's perspectives using the PEN-3 cultural model.

BACKGROUND: Access to antiretroviral therapy has increased life expectancy and survival among people living with HIV (PLWH) in African countries like Nigeria. Unfortunately, non-communicable diseases such as cardiovascular diseases are on the rise as important drivers of morbidity and mortality rates among this group. The aim of this study was to explore the perspectives of key stakeholders in Nigeria on the integration of evidence-based task-sharing strategies for hypertension care (TASSH) within existing HIV clinics in Nigeria. METHODS: Stakeholders representing PLWH, patient advocates, health care professionals (i.e. community health nurses, physicians and chief medical officers), as well as policymakers, completed in-depth qualitative interviews. Stakeholders were asked to discuss facilitators and barriers likely to influence the integration of TASSH within HIV clinics in Akwa Ibom, Nigeria. The interviews were transcribed, keywords and phrases were coded using the PEN-3 cultural model as a guide. Framework thematic analysis guided by the PEN-3 cultural model was used to identify emergent themes. RESULTS: Twenty-four stakeholders participated in the interviews. Analysis of the transcribed data using the PEN-3 cultural model as a guide yielded three emergent themes as assets for the integration of TASSH in existing HIV clinics. The themes identified are: 1) extending continuity of care among PLWH; 2) empowering health care professionals and 3) enhancing existing workflow, staff motivation, and stakeholder advocacy to strengthen the capacity of HIV clinics to integrate TASSH. CONCLUSION: These findings advance the field by providing key stakeholders with knowledge of assets within HIV clinics that can be harnessed to enhance the integration of TASSH for PLWH in Nigeria. Future studies should evaluate the effect of these assets on the implementation of TASSH within HIV clinics as well as their effect on patient-level outcomes over time.

Dissemination and implementation research coordination and training to improve cardiovascular health in people living with HIV in sub-Saharan Africa: the research coordinating center of the HLB-SIMPLe Alliance.

As global adoption of antiretroviral therapy extends the lifespan of People Living with HIV (PLHIV) through viral suppression, the risk of comorbid conditions such as hypertension has risen, creating a need for effective, scalable interventions to manage comorbidities in PLHIV. The Heart, Lung, and Blood Co-morbiditieS Implementation Models in People Living with HIV (HLB-SIMPLe) Alliance has been funded by the National Heart, Lung, and Blood Institute (NHLBI) and the Fogarty International Center (FIC) since September 2020. The Alliance was created to conduct late-stage implementation research to contextualize, implement, and evaluate evidence-based strategies to integrate the diagnosis, treatment, and control of cardiovascular diseases, particularly hypertension, in PLHIV in low- and middle-income countries (LMICs).The Alliance consists of six individually-funded clinical trial cooperative agreement research projects based in Botswana, Mozambique, Nigeria, South Africa, Uganda, and Zambia; the Research Coordinating Center; and personnel from NIH, NHLBI, and FIC (the Federal Team). The Federal Team works together with the members of the seven cooperative agreements which comprise the alliance. The Federal Team includes program officials, project scientists, grant management officials and clinical trial specialists. This Alliance of research scientists, trainees, and administrators works collaboratively to provide and support venues for ongoing information sharing within and across the clinical trials, training and capacity building in research methods, publications, data harmonization, and community engagement. The goal is to leverage shared learning to achieve collective success, where the resulting science and training are greater with an Alliance structure rather than what would be expected from isolated and unconnected individual research projects.In this manuscript, we describe how the Research Coordinating Center performs the role of providing organizational efficiencies, scientific technical assistance, research capacity building, operational coordination, and leadership to support research and training activities in this multi-project cooperative research Alliance. We outline challenges and opportunities during the initial phases of coordinating research and training in the HLB-SIMPLe Alliance, including those most relevant to dissemination and implementation researchers.

Research priorities for the primordial prevention of acute rheumatic fever and rheumatic heart disease by modifying the social determinants of health.

The social determinants of health (SDH), such as access to income, education, housing and healthcare, strongly shape the occurrence of acute rheumatic fever (ARF) and rheumatic heart disease (RHD) at the household, community and national levels. The SDH are systemic factors that privilege some more than others and result in poverty and inequitable access to resources to support health and well-being. Primordial prevention is the modification of SDH to improve health and reduce the risk of disease acquisition and the subsequent progression to RHD. Modifying these determinants using primordial prevention strategies can reduce the risk of exposure to Group A Streptococcus, a causative agent of throat and skin infections, thereby lowering the risk of initiating ARF and its subsequent progression to RHD.This report summarises the findings of the Primordial Prevention Working Group-SDH, which was convened in November 2021 by the National Heart, Lung, and Blood Institute to assess how SDH influence the risk of developing RHD. Working group members identified a series of knowledge gaps and proposed research priorities, while recognising that community engagement and partnerships with those with lived experience will be integral to the success of these activities. Specifically, members emphasised the need for: (1) global analysis of disease incidence, prevalence and SDH characteristics concurrently to inform policy and interventions, (2) global assessment of legacy primordial prevention programmes to help inform the co-design of interventions alongside affected communities, (3) research to develop, implement and evaluate scalable primordial prevention interventions in diverse settings and (4) research to improve access to and equity of services across the RHD continuum. Addressing SDH, through the implementation of primordial prevention strategies, could have broader implications, not only improving RHD-related health outcomes but also impacting other neglected diseases in low-resource settings.

Building Capacity of Community Nurses to Strengthen the Management of Uncomplicated Hypertension in Persons Living with HIV in Low- and Middle-Income Countries.

Objectives: Poor training of non-physician healthcare workers (especially community nurses) could hinder the successful integration of cardiovascular disease (CVD) management into HIV chronic care in primary healthcare facilities in low- and middle-income countries. To address this limitation, we included a holistic training programme with a robust module for both practice facilitators and community nurses as part of the formative stages of the managing hypertension among people living with HIV: an integrated model (MAP-IT), which is a study that is evaluating the effectiveness of practice facilitation on the integration of a task-strengthening strategy for hypertension control (TASSH) into primary healthcare centres in Akwa Ibom State of Nigeria. Methods: Between June and November 2021, 3 didactic training workshops were conducted using a training module which is based on the simplified Nigerian Hypertension Protocol for primary care and the World Health Organization (WHO) heart package. Knowledge acquired by the participants was assessed using anonymized pre- and post-training assessments in the first two workshops. Participants' view of the training was assessed using a comprehensive course evaluation questionnaire. Results: A total of 92 community nurses and six practice facilitators were trained in the workshops on managing hypertension in persons living with HIV. Mean pre- and post-test scores improved from 11.9(3.4) to 15.9(2.9); p < 0.001 in the first workshop, and from 15.4(0.9) to 16.4 (1.4); p < 0.001 in the second workshop. The methodology used in the training, understanding of the MAP-IT study programme, and the level of engagement was highly rated by the participants with LIKERT scores of 3.2/4.0, 3.2/4.0, and 3.1/4.0 respectively. Conclusion: Our training methodology, which involved the train-the-trainer model to deliver simplified HIV and HTN care guidelines, showed improvement in the knowledge of managing hypertension in persons living with HIV and was highly rated by participants.

Research opportunities for the primordial prevention of rheumatic fever and rheumatic heart disease-streptococcal vaccine development: a national heart, lung and blood institute workshop report.

Streptococcus pyogenes, also known as group A streptococcus (StrepA), is a bacterium that causes a range of human diseases, including pharyngitis, impetigo, invasive infections, and post-infection immune sequelae such as rheumatic fever and rheumatic heart disease. StrepA infections cause some of the highest burden of disease and death in mostly young populations in low-resource settings. Despite decades of effort, there is still no licensed StrepA vaccine, which if developed, could be a cost-effective way to reduce the incidence of disease. Several challenges, including technical and regulatory hurdles, safety concerns and a lack of investment have hindered StrepA vaccine development. Barriers to developing a StrepA vaccine must be overcome in the future by prioritising key areas of research including greater understanding of StrepA immunobiology and autoimmunity risk, better animal models that mimic human disease, expanding the StrepA vaccine pipeline and supporting vaccine clinical trials. The development of a StrepA vaccine is a complex and challenging process that requires significant resources and investment. Given the global burden of StrepA infections and the potential for a vaccine to save lives and livelihoods, StrepA vaccine development is an area of research that deserves considerable support. This report summarises the findings of the Primordial Prevention Working Group-VAX, which was convened in November 2021 by the National Heart, Lung, and Blood Institute. The focus of this report is to identify research gaps within the current StrepA vaccine landscape and find opportunities and develop priorities to promote the rapid and successful advancement of StrepA vaccines.

Organizational readiness to implement task-strengthening strategy for hypertension management among people living with HIV in Nigeria.

BACKGROUND: Hypertension (HTN) is highly prevalent among people living with HIV (PLHIV), but there is limited access to standardized HTN management strategies in public primary healthcare facilities in Nigeria. The shortage of trained healthcare providers in Nigeria is an important contributor to the increased unmet need for HTN management among PLHIV. Evidence-based TAsk-Strengthening Strategies for HTN control (TASSH) have shown promise to address this gap in other resource-constrained settings. However, little is known regarding primary health care facilities' capacity to implement this strategy. The objective of this study was to determine primary healthcare facilities' readiness to implement TASSH among PLHIV in Nigeria. METHODS: This study was conducted with purposively selected healthcare providers at fifty-nine primary healthcare facilities in Akwa-Ibom State, Nigeria. Healthcare facility readiness data were measured using the Organizational Readiness to Change Assessment (ORCA) tool. ORCA is based on the Promoting Action on Research Implementation in Health Services (PARIHS) framework that identifies evidence, context, and facilitation as the key factors for effective knowledge translation. Quantitative data were analyzed using descriptive statistics (including mean ORCA subscales). We focused on the ORCA context domain, and responses were scored on a 5-point Likert scale, with 1 corresponding to disagree strongly. FINDINGS: Fifty-nine healthcare providers (mean age 45; standard deviation [SD]: 7.4, 88% female, 68% with technical training, 56% nurses, 56% with 1-5 years providing HIV care) participated in the study. Most healthcare providers provide care to 11-30 patients living with HIV per month in their health facility, with about 42% of providers reporting that they see between 1 and 10 patients with HTN each month. Overall, staff culture (mean 4.9 [0.4]), leadership support (mean 4.9 [0.4]), and measurement/evidence-assessment (mean 4.6 [0.5]) were the topped-scored ORCA subscales, while scores on facility resources (mean 3.6 [0.8]) were the lowest. CONCLUSION: Findings show organizational support for innovation and the health providers at the participating health facilities. However, a concerted effort is needed to promote training capabilities and resources to deliver services within these primary healthcare facilities. These results are invaluable in developing future strategies to improve the integration, adoption, and sustainability of TASSH in primary healthcare facilities in Nigeria. TRIAL REGISTRATION: NCT05031819.

Study design and protocol of a stepped wedge cluster randomized trial using a practical implementation strategy as a model for hypertension-HIV integration - the MAP-IT trial.

BACKGROUND: As people living with HIV (PLWH) experience earlier and more pronounced onset of noncommunicable diseases (NCDs), advancing integrated care networks and models in low-resource-high-need settings is critical. Leveraging current health system initiatives and addressing gaps in treatment for PLWH, we report our approach using a late-stage (T4) implementation research study to test the adoption and sustainability of a proven-effective implementation strategy which has been minimally applied in low-resource settings for the integration of hypertension control into HIV treatment. We detail our protocol for the Managing Hypertension Among People Living with HIV: an Integrated Model (MAP-IT) trial, which uses a stepped wedge cluster randomized trial (SW-CRT) design to evaluate the effectiveness of practice facilitation on the adoption of a hypertension treatment program for PLWH receiving care at primary healthcare centers (PHCs) in Akwa Ibom State, Nigeria. DESIGN: In partnership with the Nigerian Federal Ministry of Health (FMOH) and community organizations, the MAP-IT trial takes place in 30 PHCs. The i-PARiHS framework guided pre-implementation needs assessment. The RE-AIM framework will guide post-implementation activities to evaluate the effect of practice facilitation on the adoption, implementation fidelity, and sustainability of a hypertension program, as well as blood pressure (BP) control. Using a SW-CRT design, PHCs sequentially crossover from the hypertension program only (usual care) to hypertension plus practice facilitation (experimental condition). PHCs will recruit and enroll an average of 28-32 patients to reach a maximum of 960 PLWH participants with uncontrolled hypertension who will be followed longitudinally for BP outcomes. DISCUSSION: Given the need for integrated NCD-HIV care platforms in low-resource settings, MAP-IT will underscore the challenges and opportunities for integrating hypertension treatment into HIV care, particularly concerning adoption and sustainability. The evaluation of our integration approach will also highlight the potential impact of a health systems strengthening approach on BP control among PLWH. TRIAL REGISTRATION: Clinicaltrials.gov ( NCT05031819 ). Registered on 2nd September 2021.

Functional Conservation of P48/45 Proteins in the Transmission Stages of Plasmodium vivax (Human Malaria Parasite) and P. berghei (Murine Malaria Parasite).

Sexual-stage proteins have a distinct function in the mosquito vector during transmission and also represent targets for the development of malaria transmission-blocking vaccine. P48/45, a leading vaccine candidate, is critical for male gamete fertility and shows >50% similarity across various species of Plasmodium We evaluated functional conservation of P48/45 in Plasmodium vivax and P. berghei with the motivation to establish transgenic P. berghei strains expressing P. vivax P48/45 (Pvs48/45) in an in vivo assay to evaluate the transmission-blocking activity of antibodies elicited by Pvs48/45. Homologous recombination was employed to target P. bergheis48/45 (pbs48/45) for knockout (KO) or for its replacement by two different forms of P. vivaxs48/45 (pvs48/45) (the full-length gene and a chimeric gene consisting of pbs48/45 5' signal and 3' anchor sequences flanking pvs48/45). Expression of Pvs48/45 in transgenic parasites and lack of expression of any P48/45 in KO parasites were confirmed by reverse transcription-PCR (RT-PCR) and Western blotting. Both transgenic and knockout parasites revealed asexual growth kinetics in mice comparable to that seen with wild-type parasites. When employed in mosquito infection experiments, both transgenic parasite strains remained transmission competent and developed into infectious sporozoites, whereas the knockout parasites were incapable of establishing mosquito-stage infection. These results indicate the functional conservation of P48/45 protein during transmission, and the transgenic parasites generated in this study represent a valuable tool to evaluate the protective efficacy of transmission-blocking antibodies elicited by Pvs48/45-based vaccines using an in vivo mouse animal assay instead of ex vivo membrane feeding assays (MFA) relying on access to P. vivax gametocytes from infected patients.IMPORTANCE Malaria transmission depends upon successful sexual differentiation and maturation of parasites in the vertebrate host and further development in the mosquito midgut. Stage-specific proteins in the sexual stages have been shown to play a critical role in development and successful transmission through the anopheline mosquito vector. Studies presented in the current manuscript evaluated functional conservation of one such protein, P48/45, in two diverse species (P. berghei and P. vivax). Replacement of endogenous pbs48/45 in P. berghei with pvs48/45 (P. vivax homologue) did not affect the viability of the parasites, and the transgenic parasites expressing Pvs48/45 remained transmission competent. These studies establish not only the functional conservation of P48/45 in P. berghei and P. vivax but also offer an opportunity to develop an in vivo test model for Pvs48/45-based P. vivax transmission-blocking vaccines, currently under development.

Antibodies elicited during natural infection in a predominantly Plasmodium falciparum transmission area cross-react with sexual stage-specific antigen in P. vivax.

Infections caused by Plasmodium falciparum and P. vivax account for more than 90% of global malaria burden. Exposure to malaria parasite elicits immune responses during natural infection and it is generally believed that the immunity is not only stage specific but also species specific. However, partial genomic similarity for various antigens in different Plasmodium spp. raises the possibility of immunological cross-reactivity at the level of specific antigens. Serum samples collected from children who were permanent residents of a P. falciparum transmission area in Zimbabwe were screened for antibody reactivity against Pfs48/45, a P. falciparum gametocyte antigen and Pvs48/45, a P. vivax homolog of Pfs48/45 using ELISA. Western blotting was used to further confirm identity of the specific antibody reactivity to the Pfs48/45 and Pvs48/45 proteins. Pan Plasmodium PCR and nested PCR were used to confirm infection with the Plasmodium species. Twenty-seven percent (49/181) of the participants were found to be sero-positive for Pfs48/45 and 73% (n=36) of these Pfs48/45 positive sera also showed reactivity with Pvs48/45. Immune cross-reactivity revealed by ELISA was also confirmed by Western blot analysis using a panel of randomly selected 23 Pfs48/45 and Pvs48/45 ELISA positive samples. Nested PCR analysis of 27 blood samples randomly selected from the 36 that showed positive ELISA reactivity to both Pfs48/45 and Pvs48/45 antigens confirmed infection with P. falciparum and generalized absence of P. vivax except for a single sample which revealed PCR positivity for both P. vivax and P. falciparum. Our studies with sera samples from a predominantly P. falciparum transmission area in Zimbabwe suggest immunological cross-reactivity with Pvs48/45, thus raising the possibility of partial species cross-reactive immunity and possible cross-boosting of immunity during co-infection with P. falciparum and P. vivax.

Immunogenicity and malaria transmission reducing potency of Pfs48/45 and Pfs25 encoded by DNA vaccines administered by intramuscular electroporation.

Pfs48/45 and Pfs25 are leading candidates for the development of Plasmodium falciparum transmission blocking vaccines (TBV). Expression of Pfs48/45 in the erythrocytic sexual stages and presentation to the immune system during infection in the human host also makes it ideal for natural boosting. However, it has been challenging to produce a fully folded, functionally active Pfs48/45, using various protein expression platforms. In this study, we demonstrate that full-length Pfs48/45 encoded by DNA plasmids is able to induce significant transmission reducing immune responses. DNA plasmids encoding Pfs48/45 based on native (WT), codon optimized (SYN), or codon optimized and mutated (MUT1 and MUT2), to prevent any asparagine (N)-linked glycosylation were compared with or without intramuscular electroporation (EP). EP significantly enhanced antibody titers and transmission blocking activity elicited by immunization with SYN Pfs48/45 DNA vaccine. Mosquito membrane feeding assays also revealed improved functional immunogenicity of SYN Pfs48/45 (N-glycosylation sites intact) as compared to MUT1 or MUT2 Pfs48/45 DNA plasmids (all N-glycosylation sites mutated). Boosting with recombinant Pfs48/45 protein after immunization with each of the different DNA vaccines resulted in significant boosting of antibody response and improved transmission reducing capabilities of all four DNA vaccines. Finally, immunization with a combination of DNA plasmids (SYN Pfs48/45 and SYN Pfs25) also provides support for the possibility of combining antigens targeting different life cycle stages in the parasite during transmission through mosquitoes.

Comparative functional potency of DNA vaccines encoding Plasmodium falciparum transmission blocking target antigens Pfs48/45 and Pfs25 administered alone or in combination by in vivo electroporation in rhesus macaques.

Antibodies recognizing conformational epitopes in Pfs48/45, an antigen expressed on the surface of Plasmodium falciparum gametes and zygotes, have firmly established Pfs48/45 as a promising transmission blocking vaccine (TBV) candidate. However, it has been difficult to reproducibly express Pfs48/45 in a variety of recombinant expression systems. The goal of our studies was to evaluate functional immunogenicity of Pfs48/45 using DNA vaccine format in rhesus macaques. An additional goal was to ensure that when used in combination with another malarial antigen, specific immunity to both antigens was not compromised. For testing combination vaccines, we employed Pfs25 DNA plasmids that have previously undergone evaluations in rodents and nonhuman primates. Pfs25 is expressed on the surface of parasites after fertilization and is also a lead TBV candidate. DNA plasmids based on codon-optimized sequences of Pfs48/45 and Pfs25 were administered by in vivo electroporation, followed by a final recombinant protein boost. Our studies demonstrate that Pfs48/45 encoded by DNA plasmids is capable of inducing potent transmission blocking antibody responses, and such transmission blocking immune potency of Pfs48/45 was not compromised when tested in combination with Pfs25, These findings provide the evidence in favor of further studies on Pfs48/45 and Pfs25, either alone or in combination with other known malaria vaccine candidates for developing effective vaccines capable of interrupting malaria transmission.

Immunological Cross-Reactivity between Malaria Vaccine Target Antigen P48/45 in Plasmodium vivax and P. falciparum and Cross-Boosting of Immune Responses.

In general, malaria immunity has been suggested to be species specific with very little, if any, known cross-reactivity between Plasmodium vivax and P. falciparum, both of which are responsible for >90% of human malaria, and co-endemic in many countries. It is therefore believed that species-specific immunity may be needed to target different species of Plasmodium. Pfs48/45 and Pvs48/45 are well established targets in the sexual stages of the malaria parasites, and are being pursued for the development of transmission blocking vaccines. Comparison of their sequences reveals 61% and 55% identity at the DNA and protein level, respectively raising the possibility that these two target antigens might share cross-reacting epitopes. Having succeeded in expressing recombinant Pfs48/45 and Pvs48/45 proteins, we hypothesized that these proteins will not only exhibit immunological cross-reactivity but also cross-boost immune responses. Mice were immunized with purified recombinant proteins using CFA, Montanide ISA-51 and alum as adjuvants, and the sera were analyzed by ELISA, Western blotting and indirect fixed and live IFA to address the hypothesis. Our studies revealed that Pvs48/45-immune sera showed strong cross-reactivity to full length Pfs48/45 protein, and the majority of this cross reactivity was in the amino-terminal and carboxyl-terminal sub-fragments of Pfs48/45. In cross-boosting experiments Pfs48/45 and Pvs48/45 antigens were able to cross-boost each other in mouse immunization studies. Additionally we also noticed an effect of adjuvants in the overall magnitude of observed cross-reactivity. These studies may have significant implications for immunity targeting transmission of both the species of malaria parasites.