Siglec-7 glyco-immune binding mAbs or NK cell engager biologics induce potent antitumor immunity against ovarian cancers.

Ovarian cancer (OC) is a lethal gynecologic malignancy, with modest responses to CPI. Engagement of additional immune arms, such as NK cells, may be of value. We focused on Siglec-7 as a surface antigen for engaging this population. Human antibodies against Siglec-7 were developed and characterized. Coculture of OC cells with PBMCs/NKs and Siglec-7 binding antibodies showed NK-mediated killing of OC lines. Anti-Siglec-7 mAb (DB7.2) enhanced survival in OC-challenged mice. In addition, the combination of DB7.2 and anti-PD-1 demonstrated further improved OC killing in vitro. To use Siglec-7 engagement as an OC-specific strategy, we engineered an NK cell engager (NKCE) to simultaneously engage NK cells through Siglec-7, and OC targets through FSHR. The NKCE demonstrated robust in vitro killing of FSHR+ OC, controlled tumors, and improved survival in OC-challenged mice. These studies support additional investigation of the Siglec-7 targeting approaches as important tools for OC and other recalcitrant cancers.

Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge.

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of TH2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased TH2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting TH1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a TH1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens.

More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions.

Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate.

Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a Plasmodium falciparum multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP119) to P. falciparum MSP8 (PfMSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP119 Here, using the PfMSP1/8 construct, we further optimized the recombinant PfMSP8 (rPfMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate Pfs25. The production of rPfs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of Pfs25 block sexual-stage development in mosquitoes. The sequence encoding mature Pfs25 was codon harmonized for expression in Escherichia coli We produced a rPfs25-PfMSP8 fusion protein [rPfs25/8(CΔS)] as well as unfused, mature rPfs25. rPfs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rPfs25/8(CΔS) was expressed and easily purified, with the Pfs25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound native Pfs25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of PfMSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.

Effects of immunosuppression on circulating adeno-associated virus capsid-specific T cells in humans.

In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4⁺ T cells, whereas numbers of circulating CD8⁺ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

A universal influenza A vaccine based on adenovirus expressing matrix-2 ectodomain and nucleoprotein protects mice from lethal challenge.

A universal influenza vaccine, designed to induce broadly cross-reactive immunity against current and future influenza A virus strains, is in critical demand to reduce the need for annual vaccinations with vaccines chosen upon predicting the predominant circulating viral strains, and to ameliorate the threat of cyclically occurring pandemics that have, in the past, killed tens of millions. Here, we describe a vaccine regimen based on sequential immunization with two serologically distinct chimpanzee-derived replication-defective adenovirus (Ad) vectors expressing the matrix-2 protein ectodomain (M2e) from three divergent strains of influenza A virus fused to the influenza virus nucleoprotein (NP) for induction of antibodies to M2e and virus-specific CD8(+) T cells to NP. In preclinical mouse models, the Ad vaccines expressing M2e and NP elicit robust NP-specific CD8(+) T-cell responses and moderate antibody responses to all three M2e sequences. Most importantly, vaccinated mice are protected against morbidity and mortality following challenge with high doses of different influenza virus strains. Protection requires both antibodies to M2e and cellular immune responses to NP.