Plasmodium Preerythrocytic Vaccine Antigens Enhance Sterile Protection in Mice Induced by Circumsporozoite Protein.

Preerythrocytic vaccines prevent malaria by targeting parasites in the clinically silent sporozoite and liver stages and preventing progression to the virulent blood stages. The leading preerythrocytic vaccine, RTS,S/AS01E (Mosquirix), entered implementation programs in 2019 and targets the major sporozoite surface antigen, circumsporozoite protein (CSP). However, in phase III clinical trials, RTS,S conferred partial protection with limited durability, indicating a need to improve CSP-based vaccination. Previously, we identified highly expressed liver-stage proteins that could potentially be used in combination with CSP; they are referred to as preerythrocytic vaccine antigens (PEVAs). Here, we developed heterologous prime-boost CSP vaccination models to confer partial sterilizing immunity against Plasmodium yoelii (protein prime-adenovirus 5 [Ad5] boost) and Plasmodium berghei (DNA prime-Ad5 boost) in mice. When combined as individual antigens with P. yoelii CSP (PyCSP), three of eight P. yoelii PEVAs significantly enhanced sterile protection against sporozoite challenge, compared to PyCSP alone. Similar results were obtained when three P. berghei PEVAs and P. berghei CSP were combined in a single vaccine regimen. In general, PyCSP antibody responses were similar after CSP alone versus CSP plus PEVA vaccinations. Both P. yoelii and P. berghei CSP plus PEVA combination vaccines induced robust CD8+ T cell responses, including signature gamma interferon (IFN-γ) increases. In the P. berghei model system, IFN-γ responses were significantly higher in hepatic versus splenic CD8+ T cells. The addition of novel antigens may enhance the degree and duration of sterile protective immunity conferred by a human vaccine such as RTS,S.

Parasite load stemming from immunization route determines the duration of liver-stage immunity.

Immunization with radiation-attenuated Plasmodium sporozoites (RAS) induces sterile and long-lasting protective immunity. Although intravenous (IV) route of RAS immunization is reported to induce superior immunity compared to intradermal (ID) injection, its role in the maintenance of sterile immunity is yet to be understood. We investigated whether the route of homologous sporozoite challenge of Plasmodium berghei (Pb) RAS-immunized mice would influence the longevity of protection. C57BL/6 mice immunized with Pb-RAS by IV were 100% protected upon primary IV/ID sporozoite challenge. In contrast, ID immunization resulted in 80% protection, regardless of primary challenge route. Interestingly, the route of primary challenge was found to bring difference in the maintenance of sterile protection. While IV Pb RAS-immunized mice remained protected at all challenges regardless of the route of primary challenge, ID Pb-RAS-immunized mice receiving ID primary challenge became parasitaemic upon secondary IV challenge. Significantly, primary IV challenge of Pb RAS ID-immunized mice resulted in 80% and 50% survival at secondary and tertiary challenges, respectively. According to phenotypically diverse liver CD8+ T cells, the percentages and the numbers of both CD8+ T effector memory and resident memory cells were significantly higher in IV than in ID Pb RAS-immunized mice. IFN-γ-producing CD8+ T cells specific to Pb TRAP130 and MIP-4-Kb-17 were also found significantly higher in IV mice than in ID mice. The enhanced T-cell generation and the longevity of protection appear to be dependent on the parasite load during challenge when infection is tolerated under suboptimal CD8+ T-cell response.

Assessment of the Plasmodium falciparum Preerythrocytic Antigen UIS3 as a Potential Candidate for a Malaria Vaccine.

Efforts are under way to improve the efficacy of subunit malaria vaccines through assessments of new adjuvants, vaccination platforms, and antigens. In this study, we further assessed the Plasmodium falciparum antigen upregulated in infective sporozoites 3 (PfUIS3) as a vaccine candidate. PfUIS3 was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) and used to immunize mice in a prime-boost regimen. We previously demonstrated that this regimen could provide partial protection against challenge with chimeric P. berghei parasites expressing PfUIS3. We now show that ChAd63-MVA PfUIS3 can also provide partial cross-species protection against challenge with wild-type P. berghei parasites. We also show that PfUIS3-specific cellular memory responses could be recalled in human volunteers exposed to P. falciparum parasites in a controlled human malaria infection study. When ChAd63-MVA PfUIS3 was coadministered with the vaccine candidate P. falciparum thrombospondin-related adhesion protein (PfTRAP) expressed in the ChAd63-MVA system, there was no significant change in immunogenicity to either vaccine. However, when mice were challenged with double chimeric P. berghei-P. falciparum parasites expressing both PfUIS3 and PfTRAP, vaccine efficacy was improved to 100% sterile protection. This synergistic effect was evident only when the two vaccines were mixed and administered at the same site. We have therefore demonstrated that vaccination with PfUIS3 can induce a consistent delay in patent parasitemia across mouse strains and against chimeric parasites expressing PfUIS3 as well as wild-type P. berghei; when this vaccine is combined with another partially protective regimen (ChAd63-MVA PfTRAP), complete protection is induced.

Frequent inoculations with radiation attenuated sporozoite is essential for inducing sterile protection that correlates with a threshold level of Plasmodia liver-stage specific CD8+ T cells.

Whole sporozoite vaccine (WSV) is shown to induce sterile protection that targets Plasmodium liver-stage infection. There are many underlying issues associated with induction of effective sterile protracted protection. In this study, we have addressed how the alterations in successive vaccine regimen could possibly affect the induction of sterile protection. We have demonstrated that the pattern of vaccination with RAS (radiation attenuated sporozoites) induces varying degrees of protection among B6 mice. Animals receiving four successive doses generated 100% sterile protection. However, three successive doses, though with the same parasite inoculum as four doses, could induce sterile protection in ∼50% mice. Interestingly, mice immunized with the same 3 doses, but with longer gap, could not survive the challenge. We demonstrate that degree of protection correlates with the frequencies of IFN-γ+ and multifunctional (IFN-γ+ CD107a+) CD8+ TEM cells present in liver. The failure to achieve protective threshold frequency of these cells in liver might make the host more vulnerable to parasite infection during infectious sporozoite challenge.

Antibody Biomarkers Associated with Sterile Protection Induced by Controlled Human Malaria Infection under Chloroquine Prophylaxis.

Immunization with sporozoites under chloroquine chemoprophylaxis (CPS) induces distinctly preerythrocytic and long-lasting sterile protection against homologous controlled human malaria infection (CHMI). To identify possible humoral immune correlates of protection, plasma samples were collected from 38 CPS-immunized Dutch volunteers for analysis using a whole Plasmodium falciparum proteome microarray with 7,455 full-length or segmented protein features displaying about 91% of the total P. falciparum proteome. We identified 548 reactive antigens representing 483 unique proteins. Using the breadth of antibody responses for each subject in a mixture-model algorithm, we observed a trimodal pattern, with distinct groups of 16 low responders, 19 medium responders, and 3 high responders. Fifteen out of 16 low responders, 12 of the 19 medium responders, and 3 out of 3 high responders were fully protected from a challenge infection. In the medium-responder group, we identified six novel antigens associated with protection (area under the curve [AUC] value of ≥0.75; P < 0.05) and six other antigens that were specifically increased in nonprotected volunteers (AUC value of ≤0.25; P < 0.05). When used in combination, the multiantigen classifier predicts CPS-induced protective efficacy with 83% sensitivity and 88% specificity. The antibody response patterns characterized in this study represent surrogate markers that may provide rational guidance for clinical vaccine development.IMPORTANCE Infection by Plasmodium parasites has been a major cause of mortality and morbidity in humans for thousands of years. Despite the considerable reduction of deaths, according to the WHO, over 5 billion people are still at risk, with about 216 million worldwide cases occurring in 2016. More compelling, 15 countries in sub-Saharan Africa bore 80% of the worldwide malaria burden. Complete eradication has been challenging, and the development of an affordable and effective vaccine will go a long way in achieving elimination. However, identifying vaccine candidate targets has been difficult. In the present study, we use a highly effective immunization protocol that confers long-lasting sterile immunity in combination with a whole P. falciparum proteome microarray to identify antibody responses associated with protection. This study characterizes a novel antibody profile associated with sterile protective immunity and trimodal humoral responses that sheds light on the possible mechanism of CPS-induced immunity against P. falciparum parasites.

Corrigendum: Liver-Stage Specific Response among Endemic Populations: Diet and Immunity.

[This corrects the article on p. 125 in vol. 6, PMID: 25852693.].

Liver-Stage Specific Response among Endemic Populations: Diet and Immunity.

Developing effective anti-malarial vaccine has been a challenge for long. Various factors including complex life cycle of parasite and lack of knowledge of stage specific critical antigens are some of the reasons. Moreover, inadequate understanding of the immune responses vis-à-vis sterile protection induced naturally by Plasmodia infection has further compounded the problem. It has been shown that people living in endemic areas take years to develop protective immunity to blood stage infection. But hardly anyone believes that immunity to liver-stage infection could be developed. Various experimental model studies using attenuated parasite suggest that liver-stage immunity might exist among endemic populations. This could be induced because of the attenuation of parasite in liver by various compounds present in the diet of endemic populations.