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.

γδ T Cells Are Required for the Induction of Sterile Immunity during Irradiated Sporozoite Vaccinations.

Whole-sporozoite vaccines confer sterilizing immunity to malaria-naive individuals by unknown mechanisms. In the first PfSPZ Vaccine trial ever in a malaria-endemic population, Vδ2 γδ T cells were significantly elevated and Vγ9/Vδ2 transcripts ranked as the most upregulated in vaccinees who were protected from Plasmodium falciparum infection. In a mouse model, absence of γδ T cells during vaccination impaired protective CD8 T cell responses and ablated sterile protection. γδ T cells were not required for circumsporozoite protein-specific Ab responses, and γδ T cell depletion before infectious challenge did not ablate protection. γδ T cells alone were insufficient to induce protection and required the presence of CD8α+ dendritic cells. In the absence of γδ T cells, CD8α+ dendritic cells did not accumulate in the livers of vaccinated mice. Altogether, our results show that γδ T cells were essential for the induction of sterile immunity during whole-organism vaccination.

Design of a stabilized RBD enables potently neutralizing SARS-CoV-2 single-component nanoparticle vaccines.

Waning immunity and emerging variants necessitate continued vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Improvements in vaccine safety, tolerability, and ease of manufacturing would benefit these efforts. Here, we develop a potent and easily manufactured nanoparticle vaccine displaying the spike receptor-binding domain (RBD). Computational design to stabilize the RBD, eliminate glycosylation, and focus the immune response to neutralizing epitopes results in an RBD immunogen that resolves issues hindering the efficient nanoparticle display of the native RBD. This non-glycosylated RBD can be genetically fused to diverse single-component nanoparticle platforms, maximizing manufacturing ease and flexibility. All engineered RBD nanoparticles elicit potently neutralizing antibodies in mice that far exceed monomeric RBDs. A 60-copy particle (noNAG-RBD-E2p) also elicits potently neutralizing antibodies in non-human primates. The neutralizing antibody titers elicited by noNAG-RBD-E2p are comparable to a benchmark stabilized spike antigen and reach levels against Omicron BA.5 that suggest that it would provide protection against emerging variants.

Aotus nancymaae model predicts human immune response to the placental malaria vaccine candidate VAR2CSA.

Placental malaria vaccines (PMVs) are being developed to prevent severe sequelae of placental malaria (PM) in pregnant women and their offspring. The leading candidate vaccine antigen VAR2CSA mediates parasite binding to placental receptor chondroitin sulfate A (CSA). Despite promising results in small animal studies, recent human trials of the first two PMV candidates (PAMVAC and PRIMVAC) generated limited cross-reactivity and cross-inhibitory activity to heterologous parasites. Here we immunized Aotus nancymaae monkeys with three PMV candidates (PAMVAC, PRIMVAC and ID1-ID2a_M1010) adjuvanted with Alhydrogel, and exploited the model to investigate boosting of functional vaccine responses during PM episodes as well as with nanoparticle antigens. PMV candidates induced high levels of antigen-specific IgG with significant cross-reactivity across PMV antigens by enzyme-linked immunosorbent assay. Conversely, PMV antibodies recognized native VAR2CSA and blocked CSA adhesion of only homologous parasites and not of heterologous parasites. PM episodes did not significantly boost VAR2CSA antibody levels or serum functional activity; nanoparticle and monomer antigens alike boosted serum reactivity but not functional activities. Overall, PMV candidates induced functional antibodies with limited heterologous activity in Aotus monkeys, similar to responses reported in humans. The Aotus model appears suitable for preclinical downselection of PMV candidates and assessment of antibody boosting by PM episodes.

Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all current vaccines. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence, and four key changes conserved between immunogens. The changes are adaptable to all vaccine platforms and compatible with mutations in emerging variants of concern. The immunogens elicit higher levels of neutralizing antibodies than native RBD, focus the immune response to structured neutralizing epitopes, and have increased production yields and thermostability. Incorporating these variant-independent amino acid changes in next-generation COVID vaccines may enhance the neutralizing antibody response and lead to longer duration and broader protection.

Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all vaccine candidates. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence and four key changes conserved between immunogens. The changes are adaptable to all vaccine platforms, are compatible with established changes in SARS-CoV-2 vaccines, and are compatible with mutations in emerging variants of concern. The immunogens elicit higher levels of neutralizing antibodies than native RBD, focus the immune response to structured neutralizing epitopes, and have increased production yields and thermostability. Incorporating these variant-independent amino acid changes in next-generation vaccines may enhance the neutralizing antibody response and lead to pan-SARS-CoV-2 protection.

Dynamics and Outcomes of Plasmodium Infections in Grammomys surdaster (Grammomys dolichurus) Thicket Rats versus Inbred Mice.

Investigations of malaria infection are often conducted by studying rodent Plasmodium species in inbred laboratory mice, but the efficacy of vaccines or adjunctive therapies observed in these models often does not translate to protection in humans. This raises concerns that mouse malaria models do not recapitulate important features of human malaria infections. African woodland thicket rats (Grammomys surdaster) are the natural host for the rodent malaria parasite Plasmodium berghei and the suspected natural host for Plasmodium vinckei vinckei. Previously, we reported that thicket rats are highly susceptible to diverse rodent parasite species, including P. berghei, Plasmodium yoelii, and Plasmodium chabaudi chabaudi, and are a more stringent model to assess the efficacy of whole-sporozoite vaccines than laboratory mice. Here, we compare the course of infection and virulence with additional rodent Plasmodium species, including various strains of P. berghei, P. yoelii, P. chabaudi, and P. vinckei, in thicket rats versus laboratory mice. We present evidence that rodent malaria parasite growth typically differs between the natural versus nonnatural host; G. surdaster limit infection by multiple rodent malaria strains, delaying and reducing peak parasitemia compared with laboratory mice. The course of malaria infection in thicket rats varied depending on parasite species and strain, resulting in self-cure, chronic parasitemia, or rapidly lethal infection, thus offering a variety of rodent malaria models to study different clinical outcomes in the natural host.

Accelerated and long term stability study of Pfs25-EPA conjugates adjuvanted with Alhydrogel®.

Pfs25, a Plasmodium falciparum surface protein expressed during zygote and ookinete stages in infected mosquitoes, is a lead transmission-blocking vaccine candidate against falciparum malaria. To enhance immunogenicity, recombinant Pfs25 was chemically conjugated to recombinant nontoxic Pseudomonas aeruginosa ExoProtein A (rEPA) in conformance with current good manufacturing practices (cGMP), and formulated with the alum adjuvant Alhydrogel. In order to meet the regulatory requirements for a phase 1 human clinical trial, the vaccine product was extensively evaluated for stability at an initial time point and through the clinical trial period annually. Because basic quality control methods to characterize alum-based vaccines remain unavailable, a thermal forced degradation study was performed prior to the initial evaluation to identify the methods suitable to detect the quality of vaccine formulations. Our results show that the vaccine product Pfs25-EPA formulated on Alhydrogel is in conformance with regulatory guidelines and suitable for human trials.

EPR oxygen images predict tumor control by a 50% tumor control radiation dose.

Clinical trials to ameliorate hypoxia as a strategy to relieve the radiation resistance it causes have prompted a need to assay the precise extent and location of hypoxia in tumors. Electron paramagnetic resonance oxygen imaging (EPR O2 imaging) provides a noninvasive means to address this need. To obtain a preclinical proof-of-principle that EPR O2 images could predict radiation control, we treated mouse tumors at or near doses required to achieve 50% control (TCD50). Mice with FSa fibrosarcoma or MCa4 carcinoma were subjected to EPR O2 imaging and immediately radiated to a TCD50 or TCD50 ± 10 Gy. Statistical analysis was permitted by collection of approximately 1,300 tumor pO2 image voxels, including the fraction of tumor voxels with pO2 less than 10 mm Hg (HF10). Tumors were followed for 90 days (FSa) or 120 days (MCa4) to determine local control or failure. HF10 obtained from EPR images showed statistically significant differences between tumors that were controlled by the TCD50 and those that were not controlled for both FSa and MCa4. Kaplan-Meier analysis of both types of tumors showed that approximately 90% of mildly hypoxic tumors were controlled (HF10%< 10%), and only 37% (FSA) and 23% (MCa4) tumors controlled if hypoxic. EPR pO2 image voxel distributions in these approximately 0.5 mL tumors provide a prediction of radiation curability independent of radiation dose. These data confirm the significance of EPR pO2 hypoxic fractions. The 90% control of low HF10 tumors argue that 0.5 mL subvolumes of tumors may be more sensitive to radiation and may need less radiation for high tumor control rates. Cancer Res; 73(17); 5328-35. ©2013 AACR.