Cellular interferon-gamma and interleukin-2 responses to SARS-CoV-2 structural proteins are broader and higher in those vaccinated after SARS-CoV-2 infection compared to vaccinees without prior SARS-CoV-2 infection.

Class I- and Class II-restricted epitopes have been identified across the SARS-CoV-2 structural proteome. Vaccine-induced and post-infection SARS-CoV-2 T-cell responses are associated with COVID-19 recovery and protection, but the precise role of T-cell responses remains unclear, and how post-infection vaccination ('hybrid immunity') further augments this immunity To accomplish these goals, we studied healthy adult healthcare workers who were (a) uninfected and unvaccinated (n = 12), (b) uninfected and vaccinated with Pfizer-BioNTech BNT162b2 vaccine (2 doses n = 177, one dose n = 1) or Moderna mRNA-1273 vaccine (one dose, n = 1), and (c) previously infected with SARS-CoV-2 and vaccinated (BNT162b2, two doses, n = 6, one dose n = 1; mRNA-1273 two doses, n = 1). Infection status was determined by repeated PCR testing of participants. We used FluoroSpot Interferon-gamma (IFN-γ) and Interleukin-2 (IL-2) assays, using subpools of 15-mer peptides covering the S (10 subpools), N (4 subpools) and M (2 subpools) proteins. Responses were expressed as frequencies (percent positive responders) and magnitudes (spot forming cells/106 cytokine-producing peripheral blood mononuclear cells [PBMCs]). Almost all vaccinated participants with no prior infection exhibited IFN-γ, IL-2 and IFN-γ+IL2 responses to S glycoprotein subpools (89%, 93% and 27%, respectively) mainly directed to the S2 subunit and were more robust than responses to the N or M subpools. However, in previously infected and vaccinated participants IFN-γ, IL-2 and IFN-γ+IL2 responses to S subpools (100%, 100%, 88%) were substantially higher than vaccinated participants with no prior infection and were broader and directed against nine of the 10 S glycoprotein subpools spanning the S1 and S2 subunits, and all the N and M subpools. 50% of uninfected and unvaccinated individuals had IFN-γ but not IL2 or IFN-γ+IL2 responses against one S and one M subpools that were not increased after vaccination of uninfected or SARS-CoV-2-infected participants. Summed IFN-γ, IL-2, and IFN-γ+IL2 responses to S correlated with IgG responses to the S glycoprotein. These studies demonstrated that vaccinations with BNT162b2 or mRNA-1273 results in T cell-specific responses primarily against epitopes in the S2 subunit of the S glycoprotein, and that individuals that are vaccinated after SARS-CoV-2 infection develop broader and greater T cell responses to S1 and S2 subunits as well as the N and M proteins.

CHARM: COVID-19 Health Action Response for Marines-Association of antigen-specific interferon-gamma and IL2 responses with asymptomatic and symptomatic infections after a positive qPCR SARS-CoV-2 test.

SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses against S, N and M proteins in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were enrolled in an immune response sub-study. FluoroSpot interferon-gamma (IFN-γ) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-γ responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-γ responses were significantly (p = 0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic but not symptomatic subjects. Asymptomatic IFN-γ responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but not symptomatic participants. The frequencies of IFN-γ responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-γ and IL2 responses to the N+M pool were significantly correlated with IFN-γ and IL2 responses to the N and M pools. These data further support the central role of Th1-biased cell mediated immunity IFN-γ and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.

A three-antigen Plasmodium falciparum DNA prime-Adenovirus boost malaria vaccine regimen is superior to a two-antigen regimen and protects against controlled human malaria infection in healthy malaria-naïve adults.

BACKGROUND: A DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen [malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP)] to potentially enhance protection. METHODOLOGY: This was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method. RESULTS: In both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-γ FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection. CONCLUSIONS: This study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.

IMRAS-Immunization with radiation-attenuated Plasmodium falciparum sporozoites by mosquito bite: Cellular immunity to sporozoites, CSP, AMA1, TRAP and CelTOS.

BACKGROUND: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bites provides >90% sterile protection against Plasmodium falciparum malaria in humans. We conducted a clinical trial based on data from previous RAS clinical trials that suggested that 800-1200 infected bites should induce ~50% protective vaccine efficacy (VE) against controlled human malaria infection (CHMI) administered three weeks after the final immunization. Two cohorts were immunized separately. VE was 55% in Cohort 1 but 90% in Cohort 2, the cohort that received a higher first dose and a reduced (fractional) fifth dose. Immune responses were better boosted by the fractional fifth dose in Cohort 2 and suggested the importance of the fractional fifth dose for increased protection in Cohort 2 responses. Three protected subjects were later boosted and were protected suggesting that protection could be extended to at least 67 weeks. METHODS: The ex vivo FluoroSpot assay was used to measure peripheral IFN-γ, IL2, and IFN-γ+IL2 responses to PfNF54 sporozoites and malaria antigens CSP, AMA1, TRAP, and CelTOS using pools of synthetic overlapping 15mer peptides spanning each antigen. RESULTS: There was no correlation between IFN-γ, IL2, and IFN-γ+IL2 responses to sporozoites and protection, but fold-increases between post-4th and post-5th responses greater than 1.0 occurred mostly in protected subjects. IFN-γ and IL2 responses to TRAP, CelTOS and CSP occurred only in protected subjects. Peripheral IFN-γ, IL2, and IFN-γ+IL2 responses were short-lived and low by 27 weeks post-CHMI but were restored by boosting. CONCLUSIONS: These studies highlight the importance of vaccine dose and schedule for vaccine efficacy, and suggest that CSP, TRAP, AMA1 and CelTOS may be targets of protective immunity. The correlation between fold-increases in responses and protection should be explored in other vaccine trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT01994525.

IMRAS-A clinical trial of mosquito-bite immunization with live, radiation-attenuated P. falciparum sporozoites: Impact of immunization parameters on protective efficacy and generation of a repository of immunologic reagents.

BACKGROUND: Immunization with radiation-attenuated sporozoites (RAS) by mosquito bite provides >90% sterile protection against Plasmodium falciparum (Pf) malaria in humans. RAS invade hepatocytes but do not replicate. CD8+ T cells recognizing parasite-derived peptides on the surface of infected hepatocytes are likely the primary protective mechanism. We conducted a randomized clinical trial of RAS immunization to assess safety, to achieve 50% vaccine efficacy (VE) against controlled human malaria infection (CHMI), and to generate reagents from protected and non-protected subjects for future identification of protective immune mechanisms and antigens. METHODS: Two cohorts (Cohort 1 and Cohort 2) of healthy, malaria-naïve, non-pregnant adults age 18-50 received five monthly immunizations with infected (true-immunized, n = 21) or non-infected (mock-immunized, n = 5) mosquito bites and underwent homologous CHMI at 3 weeks. Immunization parameters were selected for 50% protection based on prior clinical data. Leukapheresis was done to collect plasma and peripheral blood mononuclear cells. RESULTS: Adverse event rates were similar in true- and mock-immunized subjects. Two true- and two mock-immunized subjects developed large local reactions likely caused by mosquito salivary gland antigens. In Cohort 1, 11 subjects received 810-1235 infected bites; 6/11 (55%) were protected against CHMI vs. 0/3 mock-immunized and 0/6 infectivity controls (VE 55%). In Cohort 2, 10 subjects received 839-1131 infected bites with a higher first dose and a reduced fifth dose; 9/10 (90%) were protected vs. 0/2 mock-immunized and 0/6 controls (VE 90%). Three/3 (100%) protected subjects administered three booster immunizations were protected against repeat CHMI vs. 0/6 controls (VE 100%). Cohort 2 uniquely showed a significant rise in IFN-γ responses after the third and fifth immunizations and higher antibody responses to CSP. CONCLUSIONS: PfRAS were generally safe and well tolerated. Cohort 2 had a higher first dose, reduced final dose, higher antibody responses to CSP and significant rise of IFN-γ responses after the third and fifth immunizations. Whether any of these factors contributed to increased protection in Cohort 2 requires further investigation. A cryobank of sera and cells from protected and non-protected individuals was generated for future immunological studies and antigen discovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01994525.

Novel malaria antigen Plasmodium yoelii E140 induces antibody-mediated sterile protection in mice against malaria challenge.

Only a small fraction of the antigens expressed by malaria parasites have been evaluated as vaccine candidates. A successful malaria subunit vaccine will likely require multiple antigenic targets to achieve broad protection with high protective efficacy. Here we describe protective efficacy of a novel antigen, Plasmodium yoelii (Py) E140 (PyE140), evaluated against P. yoelii challenge of mice. Vaccines targeting PyE140 reproducibly induced up to 100% sterile protection in both inbred and outbred murine challenge models. Although PyE140 immunization induced high frequency and multifunctional CD8+ T cell responses, as well as CD4+ T cell responses, protection was mediated by PyE140 antibodies acting against blood stage parasites. Protection in mice was long-lasting with up to 100% sterile protection at twelve weeks post-immunization and durable high titer anti-PyE140 antibodies. The E140 antigen is expressed in all Plasmodium species, is highly conserved in both P. falciparum lab-adapted strains and endemic circulating parasites, and is thus a promising lead vaccine candidate for future evaluation against human malaria parasite species.

New gorilla adenovirus vaccine vectors induce potent immune responses and protection in a mouse malaria model.

BACKGROUND: A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors. RESULTS: The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen. CONCLUSION: These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

Cellular immune response to DNA and vaccinia prime-boost immunization kills Plasmodium yoelii-infected hepatocytes in vitro.

Background: Plasmid DNA encoding Plasmodium yoelii circumsporozoite protein (PyCSP) followed by boosting with recombinant vaccinia virus containing the PyCSP elicited significant protective immunity in mice that was primarily mediated by CD8+ T-cell responses directed to P. yoelii -infected hepatocytes. This study was to further explore protection using in vitro cultures of P. yoelii parasites in mouse hepatocytes. Spleen cells from DNA/vaccinia virus-immunized mice were co-cultured in vitro with mouse hepatocytes containing developing P. yoelii liver stage parasites. A semipermeable membrane separating spleen cells and hepatocytes was used to demonstrate if cell-to-cell contact was required. Inhibitors of mediators likely involved in spleen cell killing were added to these co-cultures. Spleen cells from immunized mice inhibited in vitro P. yoelii parasite development, and inhibition was eliminated by separating effectors and targets with the semipermeable membrane. Additionally, inhibitors of inducible nitric oxide synthase, caspase activation, NF-κB activation as well as antibodies against interferon-gamma (IFN-γ) and ICAM-1 reduced parasite inhibition. These findings suggest that direct contact between spleen cells from immunized mice and P. yoelii-infected hepatocytes is required for eliminating liver stage parasites and provide more insight into CD8+ T-cell-mediated inhibition of malaria liver stage development.

Development of whole sporozoite malaria vaccines.

INTRODUCTION: Despite recent advances, malaria remains a major health threat both to populations in endemic areas as well travelers, including military personnel, to these areas. Subunit vaccines have not yet achieved sufficient efficacy needed for use in any of these at risk populations. Areas covered: This review discusses the current status of various whole sporozoite vaccine approaches and is mainly focused on current clinical trials. Expert commentary: Nearly 100% efficacy was achieved by administering multiple bites of radiation-attenuated sporozoite (RAS) Plasmodium falciparum-infected mosquitoes; this is impractical for widespread use. Now, this high level efficacy has been reproduced using purified, metabolically active RAS (PfSPZ Sanaria® Vaccine), which is undergoing extensive clinical testing. Alternative whole sporozoite vaccines include immunization with fully infectious sporozoites under chloroquine prophylaxis (CPS) or as genetically-attenuated parasites (GAP). By also manufacturing purified infectious sporozoites, it is now possible to combine these with CPS and GAP, as well as perform challenge studies using controlled doses of sporozoites.

Development of replication-deficient adenovirus malaria vaccines.

INTRODUCTION: Malaria remains a major threat to endemic populations and travelers, including military personnel to these areas. A malaria vaccine is feasible, as radiation attenuated sporozoites induce nearly 100% efficacy. Areas covered: This review covers current malaria clinical trials using adenoviruses and pre-clinical research. Heterologous prime-boost regimens, including replication-deficient human adenovirus 5 (HuAd5) carrying malaria antigens, are efficacious. However, efficacy appears to be adversely affected by pre-existing anti-HuAd5 antibodies. Current strategies focus on replacing HuAd5 with rarer human adenoviruses or adenoviruses isolated from non-human primates (NHPs). The chimpanzee adenovirus ChAd63 is undergoing evaluation in clinical trials including infants in malaria-endemic areas. Key antigens have been identified and are being used alone, in combination, or with protein subunit vaccines. Gorilla adenoviruses carrying malaria antigens are also currently being evaluated in preclinical models. These replacement adenovirus vectors will be successfully used to develop vaccines against malaria, as well as other infectious diseases. Expert commentary: Simplified prime-boost single shot regimens, dry-coated live vector vaccines or silicon microneedle arrays could be developed for malaria or other vaccines. Replacement vectors with similar or superior immunogenicity have rapidly advanced, and several are now in extensive Phase 2 and beyond in malaria as well as other diseases, notably Ebola.

Correction: Vaccine Strain-Specificity of Protective HLA-Restricted Class 1 P. falciparum Epitopes.

[This corrects the article DOI: 10.1371/journal.pone.0163026.].

Vaccine Strain-Specificity of Protective HLA-Restricted Class 1 P. falciparum Epitopes.

A DNA prime/adenovirus boost malaria vaccine encoding Plasmodium falciparum strain 3D7 CSP and AMA1 elicited sterile clinical protection associated with CD8+ T cell interferon-gamma (IFN-γ) cells responses directed to HLA class 1-restricted AMA1 epitopes of the vaccine strain 3D7. Since a highly effective malaria vaccine must be broadly protective against multiple P. falciparum strains, we compared these AMA1 epitopes of two P. falciparum strains (7G8 and 3D7), which differ by single amino acid substitutions, in their ability to recall CD8+ T cell activities using ELISpot and flow cytometry/intracellular staining assays. The 7G8 variant peptides did not recall 3D7 vaccine-induced CD8+ T IFN-γ cell responses in these assays, suggesting that protection may be limited to the vaccine strain. The predicted MHC binding affinities of the 7G8 variant epitopes were similar to the 3D7 epitopes, suggesting that the amino acid substitutions of the 7G8 variants may have interfered with TCR recognition of the MHC:peptide complex or that the 7G8 variant may have acted as an altered peptide ligand. These results stress the importance of functional assays in defining protective epitopes. Clinical Trials Registrations: NCT00870987, NCT00392015.

Mosquito bite immunization with radiation-attenuated Plasmodium falciparum sporozoites: safety, tolerability, protective efficacy and humoral immunogenicity.

BACKGROUND: In this phase 1 clinical trial, healthy adult, malaria-naïve subjects were immunized with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) by mosquito bite and then underwent controlled human malaria infection (CHMI). The PfRAS model for immunization against malaria had previously induced >90 % sterile protection against homologous CHMI. This study was to further explore the safety, tolerability and protective efficacy of the PfRAS model and to provide biological specimens to characterize protective immune responses and identify protective antigens in support of malaria vaccine development. METHODS: Fifty-seven subjects were screened, 41 enrolled and 30 received at least one immunization. The true-immunized subjects received PfRAS via mosquito bite and the mock-immunized subjects received mosquito bites from irradiated uninfected mosquitoes. Sera and peripheral blood mononuclear cells (PBMCs) were collected before and after PfRAS immunizations. RESULTS: Immunization with PfRAS was generally safe and well tolerated, and repeated immunization via mosquito bite did not appear to increase the risk or severity of AEs. Local adverse events (AEs) of true-immunized and mock-immunized groups consisted of erythaema, papules, swelling, and induration and were consistent with reactions from mosquito bites seen in nature. Two subjects, one true- and one mock-immunized, developed large local reactions that completely resolved, were likely a result of mosquito salivary antigens, and were withdrawn from further participation as a safety precaution. Systemic AEs were generally rare and mild, consisting of headache, myalgia, nausea, and low-grade fevers. Two true-immunized subjects experienced fever, malaise, myalgia, nausea, and rigours approximately 16 h after immunization. These symptoms likely resulted from pre-formed antibodies interacting with mosquito salivary antigens. Ten subjects immunized with PfRAS underwent CHMI and five subjects (50 %) were sterilely protected and there was a significant delay to parasitaemia in the other five subjects. All ten subjects developed humoral immune responses to whole sporozoites and to the circumsporozoite protein prior to CHMI, although the differences between protected and non-protected subjects were not statistically significant for this small sample size. CONCLUSIONS: The protective efficacy of this clinical trial (50 %) was notably less than previously reported (>90 %). This may be related to differences in host genetics or the inherent variability in mosquito biting behavior and numbers of sporozoites injected. Differences in trial procedures, such as the use of leukapheresis prior to CHMI and of a longer interval between the final immunization and CHMI in these subjects compared to earlier trials, may also have reduced protective efficacy. This trial has been retrospectively registered at ISRCTN ID 17372582, May 31, 2016.

Measurement of ex vivo ELISpot interferon-gamma recall responses to Plasmodium falciparum AMA1 and CSP in Ghanaian adults with natural exposure to malaria.

BACKGROUND: Malaria eradication requires a concerted approach involving all available control tools, and an effective vaccine would complement these efforts. An effective malaria vaccine should be able to induce protective immune responses in a genetically diverse population. Identification of immunodominant T cell epitopes will assist in determining if candidate vaccines will be immunogenic in malaria-endemic areas. This study therefore investigated whether class I-restricted T cell epitopes of two leading malaria vaccine antigens, Plasmodium falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), could recall T cell interferon-γ responses from naturally exposed subjects using ex vivo ELISpot assays. METHODS: Thirty-five subjects aged between 24 and 43 years were recruited from a malaria-endemic urban community of Ghana in 2011, and their peripheral blood mononuclear cells (PBMCs) were tested in ELISpot IFN-γ assays against overlapping 15mer peptide pools spanning the entire CSP and AMA1 antigens, and 9-10mer peptide epitope mixtures that included previously identified and/or predicted human leukocyte antigen (HLA) class 1-restricted epitopes from same two antigens. RESULTS: For CSP, 26 % of subjects responded to at least one of the nine 15mer peptide pools whilst 17 % responded to at least one of the five 9-10mer HLA-restricted epitope mixtures. For AMA1, 63 % of subjects responded to at least one of the 12 AMA1 15mer peptide pools and 51 % responded to at least one of the six 9-10mer HLA-restricted epitope mixtures. Following analysis of data from the two sets of peptide pools, along with bioinformatics predictions of class I-restricted epitopes and the HLA supertypes expressed by a subset of study subjects, peptide pools that may contain epitopes recognized by multiple HLA supertypes were identified. Collectively, these results suggest that natural transmission elicits ELISpot IFN-γ activities to class 1-restricted epitopes that are largely HLA-promiscuous. CONCLUSIONS: These results generally demonstrate that CSP and AMA1 peptides recalled ELISpot IFN-γ responses from naturally exposed individuals and that both CSP and AMA1 contain diverse class 1-restricted epitopes that are HLA-promiscuous and are widely recognized in this population.

Controlled Human Malaria Infection (CHMI) differentially affects cell-mediated and antibody responses to CSP and AMA1 induced by adenovirus vaccines with and without DNA-priming.

We have previously shown that a DNA-prime followed by an adenovirus-5 boost vaccine containing CSP and AMA1 (DNA/Ad) successfully protected 4 of 15 subjects to controlled human malaria infection (CHMI). However, the adenovirus-5 vaccine alone (AdCA) failed to induce protection despite eliciting cellular responses that were often higher than those induced by DNA/Ad. Here we determined the effect of CHMI on pre-CHMI cellular and antibody responses against CSP and AMA1 expressed as fold-changes in activities. Generally, in the DNA/Ad trial, CHMI caused pre-CHMI ELISpot IFN-γ and CD8+ T cell IFN-γ responses of the protected subjects to fall but among non-protected subjects, CHMI caused rises of pre-CHMI ELISpot IFN-γ but falls of CD8+ T cell IFN-γ responses. In contrast in the AdCA trial, CHMI caused both pre-CHMI ELISpot IFN-γ and CD8+ T cell IFN-γ responses of the AdCA subjects to fall. We suggest that the falls in activities are due to migration of peripheral CD8+ T cells to the liver in response to developing liver stage parasites, and this fall, in the DNA/Ad trial, is masked in ELISpot responses of the non-protected subjects by rises in other immune cell types. In addition, CHMI caused falls in antibody activities of protected subjects, but rises in non-protected subjects in both trials to CSP, and dramatically in the AdCA trial to AMA1, reaching 380 µg/ml that is probably due to boosting by transient blood stage infection before chloroquine treatment. Taken together, these results further define differences in cellular responses between DNA/Ad and AdCA trials, and suggest that natural transmission may boost responses induced by these malaria vaccines especially when protection is not achieved.

Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development.

BACKGROUND: Nearly 100% protection against malaria infection can be achieved in humans by immunization with P. falciparum radiation-attenuated sporozoites (RAS). Although it is thought that protection is mediated by T cell and antibody responses, only a few of the many pre-erythrocytic (sporozoite and liver stage) antigens that are targeted by these responses have been identified. METHODOLOGY: Twenty seven P. falciparum pre-erythrocytic antigens were selected using bioinformatics analysis and expression databases and were expressed in a wheat germ cell-free protein expression system. Recombinant proteins were recognized by plasma from RAS-immunized subjects, and 21 induced detectable antibody responses in mice and rabbit and sera from these immunized animals were used to characterize these antigens. All 21 proteins localized to the sporozoite: five localized to the surface, seven localized to the micronemes, cytoplasm, endoplasmic reticulum or nucleus, two localized to the surface and cytoplasm, and seven remain undetermined. PBMC from RAS-immunized volunteers elicited positive ex vivo or cultured ELISpot responses against peptides from 20 of the 21 antigens. CONCLUSIONS: These T cell and antibody responses support our approach of using reagents from RAS-immunized subjects to screen potential vaccine antigens, and have led to the identification of a panel of novel P. falciparum antigens. These results provide evidence to further evaluate these antigens as vaccine candidates. TRIAL REGISTRATION: ClinicalTrials.gov NCT00870987 ClinicalTrials.gov NCT00392015.

Measuring naturally acquired ex vivo IFN-γ responses to Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (CelTOS) in Ghanaian adults.

BACKGROUND: A malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Plasmodium falciparum. METHODS: Ex vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools (CelTp1, CelTp2, CelTp3 and CelTp4), a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control. RESULTS: Of the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP. CONCLUSIONS: These results suggest natural malaria transmission induces CelTOS-specific ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.

Sterile immunity to malaria after DNA prime/adenovirus boost immunization is associated with effector memory CD8+T cells targeting AMA1 class I epitopes.

BACKGROUND: Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection. METHODOLOGY/PRINCIPAL FINDINGS: We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. CONCLUSIONS/SIGNIFICANCE: We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.

Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection.

BACKGROUND: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial. METHODOLOGY/PRINCIPAL FINDINGS: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 10 (10) particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range<50-1626) and AMA1 ELISA of 4.95 µg/mL (range 0.2-38). Summed ex vivo IFN-γ ELISpot responses to overlapping peptides were robust, with geometric mean spot forming cells/million peripheral blood mononuclear cells [sfc/m] for CSP of 273 (range 38-2550) and for AMA1 of 1303 (range 435-4594). CD4+ and CD8+ T cell IFN-γ responses to CSP were positive by flow cytometry in 25% and 56% of the research subjects, respectively, and to AMA1 in 94% and 100%, respectively. SIGNIFICANCE: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015.

Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP).

BACKGROUND: Plasmodium falciparum circumsporozoite protein (CSP) is a leading malaria vaccine candidate antigen, known to elicit protective antibody responses in humans (RTS,S vaccine). Recently, a DNA prime / adenovirus (Ad) vector boost vaccine encoding CSP and a second P. falciparum antigen, apical membrane antigen-1, also elicited sterile protection, but in this case associated with interferon gamma ELISpot and CD8+ T cell but not antibody responses. The finding that CSP delivered by an appropriate vaccine platform likely elicits protective cell-mediated immunity provided a rationale for identifying class I-restricted epitopes within this leading vaccine candidate antigen. METHODS: Limited samples of peripheral blood mononuclear cells from clinical trials of the Ad vaccine were used to identify CD8+ T cell epitopes within pools of overlapping 15mer peptides spanning portions of CSP that stimulated recall responses. Computerized algorithms (NetMHC) predicted 17 minimal class I-restricted 9-10mer epitopes within fifteen 15mers positive in ELISpot assay using PBMC from 10 HLA-matched study subjects. Four additional epitopes were subsequently predicted using NetMHC, matched to other study subjects without initial 15mer ELISpot screening. Nine of the putative epitopes were synthesized and tested by ELISpot assay, and six of these nine were further tested for CD8+ T cell responses by ELISpot CD4+ and CD8+ T cell-depletion and flow cytometry assays for evidence of CD8+ T cell dependence. RESULTS: Each of the nine putative epitopes, all sequence-conserved, recalled responses from HLA-matched CSP-immunized research subjects. Four shorter sequences contained within these sequences were identified using NetMHC predictions and may have contributed to recall responses. Five (9-10mer) epitopes were confirmed to be targets of CD8+ T cell responses using ELISpot depletion and ICS assays. Two 9mers among these nine epitopes were each restricted by two HLA supertypes (A01/B07; A01A24/A24) and one 9mer was restricted by three HLA supertypes (A01A24/A24/B27) indicating that some CSP class I-restricted epitopes, like DR epitopes, may be HLA-promiscuous. CONCLUSIONS: This study identified nine and confirmed five novel class I epitopes restricted by six HLA supertypes, suggesting that an adenovirus-vectored CSP vaccine would be immunogenic and potentially protective in genetically diverse populations.