Immunogenicity and Efficacy of TNX-1800, A Live Virus Recombinant Poxvirus Vaccine Candidate, against SARS-CoV-2 Challenge in Nonhuman Primates.

TNX-1800 is a synthetically derived live recombinant chimeric horsepox virus (rcHPXV) vaccine candidate expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated with no serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding of anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In addition, a single dose of TNX-1800 induced an interferon-gamma (IFN-γ)-mediated T-cell response in Cynomolgus Macaques. Following challenge with SARS-CoV-2, African Green and Cynomolgus Macaques exhibited rapid clearance of virus in the upper and lower respiratory tract. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.

Single Dose of Recombinant Chimeric Horsepox Virus (TNX-801) Vaccination Protects Macaques from Lethal Monkeypox Challenge.

The ongoing global Monkeypox outbreak that started in the spring of 2022 has reinforced the importance of protecting the population using live virus vaccines based on the vaccinia virus (VACV). Smallpox also remains a biothreat and although some U.S. military personnel are immunized with VACV, safety concerns limit its use in other vulnerable groups. Consequently, there is a need for an effective and safer, single dose, live replicating vaccine against both viruses. One potential approach is to use the horsepox virus (HPXV) as a vaccine. Contemporary VACV shares a common ancestor with HPXV, which from the time of Edward Jenner and through the 19th century, was extensively used to vaccinate against smallpox. However, it is unknown if early HPXV-based vaccines exhibited different safety and efficacy profiles compared to modern VACV. A deeper understanding of HPXV as a vaccine platform may allow the construction of safer and more effective vaccines against the poxvirus family. In a proof-of-concept study, we vaccinated cynomolgus macaques with TNX-801, a recombinant chimeric horsepox virus (rcHPXV), and showed that the vaccine elicited protective immune responses against a lethal challenge with monkeypox virus (MPXV), strain Zaire. The vaccine was well tolerated and protected animals from the development of lesions and severe disease. These encouraging data support the further development of TNX-801.

Neurovirulence, viscerotropism and immunogenicity of live attenuated yellow fever 17D vaccine virus in non-human primates.

Yellow fever vaccine associated neurovirulence and viscerotropism have been reported by various countries. In this study, the neurovirulence, viscerotropism and immunogenicity of yellow fever vaccine seed lots (master and working) and final product manufactured at Serum Institute of India (SII) were evaluated in cynomolgus monkeys. WHO reference virus 168-73 and Stamaril™ as a control vaccine was used for comparison. Neurovirulence and viscerotropism scores of the seed lots and final product were lower than Stamaril™. The SII seed virus and vaccine complies to the WHO requirement for neurovirulence, viscerotropism and immunogenicity, when tested in comparison to WHO reference seed virus 168/73. All challenged animals showed 100 % seroconversion as early as day 14 and neutralizing antibody titers were sustainable at day 30 in all animals.

SARS-CoV-2 Spike Pseudoviruses: A Useful Tool to Study Virus Entry and Address Emerging Neutralization Escape Phenotypes.

SARS-CoV-2 genetic variants are emerging around the globe. Unfortunately, several SARS-CoV-2 variants, especially variants of concern (VOCs), are less susceptible to neutralization by the convalescent and post-vaccination sera, raising concerns of increased disease transmissibility and severity. Recent data suggests that SARS-CoV-2 neutralizing antibody levels are a reliable correlate of vaccine-mediated protection. However, currently used BSL3-based virus micro-neutralization (MN) assays are more laborious, time-consuming, and expensive, underscoring the need for BSL2-based, cost-effective neutralization assays against SARS-CoV-2 variants. In light of this unmet need, we have developed a BSL-2 pseudovirus-based neutralization assay (PBNA) in cells expressing the human angiotensin-converting enzyme-2 (hACE2) receptor for SARS-CoV-2. The assay is reproducible (R2 = 0.96), demonstrates a good dynamic range and high sensitivity. Our data suggest that the biological Anti-SARS-CoV-2 research reagents such as NIBSC 20/130 show lower neutralization against B.1.351 SA (South Africa) and B.1.1.7 UK (United Kingdom) VOC, whereas a commercially available monoclonal antibody MM43 retains activity against both these variants. SARS-CoV-2 spike PBNAs for VOCs would be useful tools to measure the neutralization ability of candidate vaccines in both preclinical models and clinical trials and would further help develop effective prophylactic countermeasures against emerging neutralization escape phenotypes.

Changes in the gut microbiome community of nonhuman primates following radiation injury.

BACKGROUND: Composition and maintenance of the microbiome is vital to gut homeostasis. However, there is limited knowledge regarding the impact of high doses of radiation, which can occur as a result of cancer radiation therapy, nuclear accidents or intentional release of a nuclear or radioactive weapon, on the composition of the gut microbiome. Therefore, we sought to analyze alterations to the gut microbiome of nonhuman primates (NHPs) exposed to high doses of radiation. Fecal samples were collected from 19 NHPs (Chinese rhesus macaques, Macaca mulatta) 1 day prior and 1 and 4 days after exposure to 7.4 Gy cobalt-60 gamma-radiation (LD70-80/60). The 16S V4 rRNA sequences were extracted from each sample, followed by bioinformatics analysis using the QIIME platform. RESULTS: Alpha Diversity (Shannon Diversity Index), revealed no major difference between pre- and post-irradiation, whereas Beta diversity analysis showed significant differences in the microbiome after irradiation (day + 4) compared to baseline (pre-irradiation). The Firmicutes/Bacteriodetes ratio, a factor known to be associated with disruption of metabolic homeostasis, decreased from 1.2 to less than 1 post-radiation exposure. Actinobacillus, Bacteroides, Prevotella (Paraprevotellaceae family) and Veillonella genera were significantly increased by more than 2-fold and Acinetobacter and Aerococcus genus were decreased by more than 10-fold post-irradiation. Fifty-two percent (10/19) of animals exposed to radiation demonstrated diarrhea at day 4 post-irradiation. Comparison of microbiome composition of feces from animals with and without diarrhea at day 4 post-irradiation revealed an increase in Lactobacillus reuteri associated with diarrhea and a decrease of Lentisphaerae and Verrucomicrobioa phyla and Bacteroides in animals exhibiting diarrhea. Animals with diarrhea at day 4 post-irradiation, had significantly lower levels of Lentisphaere and Verrucomicrobia phyla and Bacteroides genus at baseline before irradiation, suggesting a potential association between the prevalence of microbiomes and differential susceptibility to radiation-induced diarrhea. CONCLUSIONS: Our findings demonstrate that substantial alterations in the microbiome composition of NHPs occur following radiation injury and provide insight into early changes with high-dose, whole-body radiation exposure. Future studies will help identify microbiome biomarkers of radiation exposure and develop effective therapeutic intervention to mitigate the radiation injury.

Well-balanced immune response and protective efficacy induced by a single dose of live attenuated tetravalent dengue vaccine (KD-382) in monkeys.

One of the challenges developing a live attenuated tetravalent dengue vaccine (TDV) is to overcome the presumed viral interference that may be preventing the induction of a balanced immune response to all 4 serotypes of the dengue virus (DENV1-4). Our live attenuated TDV candidate was developed from wild-type (wt) parental strains (DENV1/03135, DENV2/99345, DENV3/16562, and DENV4/1036, respectively) using a classical host range mutation strategy: the same strategy used for the approved live attenuated smallpox, polio, and MMR vaccines. Our vaccine candidate is expected to mimic natural dengue virus infection, as it provides all the components of dengue virus, including both structural and nonstructural proteins. Therefore, induction of more solid and comprehensive immune responses against pathogenic dengue viruses is also expected. In this study, we evaluated the neutralizing antibody responses for each serotype induced by a single subcutaneous administration of 6 formulations, which were composed of different combinations of vaccine strains and were all of different dosages. These formulations were tested in dengue-naïve cynomolgus macaques. As a result, regardless of the TDV formulation, all the monkeys immunized with TDVs seroconverted to all the 4 serotypes at day 30. Next, we evaluated protection ability of the selected formulations of TDV candidate, no RNAemia was detected from any of the immunized monkeys upon s.c. challenge with wtDENV. The findings of this non-human primate study indicate that our vaccine candidate is very promising; it can be further evaluated for safety and efficacy in human clinical studies.

The vaccinia virus based Sementis Copenhagen Vector vaccine against Zika and chikungunya is immunogenic in non-human primates.

The Sementis Copenhagen Vector (SCV) is a new vaccinia virus-derived, multiplication-defective, vaccine technology assessed herein in non-human primates. Indian rhesus macaques (Macaca mulatta) were vaccinated with a multi-pathogen recombinant SCV vaccine encoding the structural polyproteins of both Zika virus (ZIKV) and chikungunya virus (CHIKV). After one vaccination, neutralising antibody responses to ZIKV and four strains of CHIKV, representative of distinct viral genotypes, were generated. A second vaccination resulted in significant boosting of neutralising antibody responses to ZIKV and CHIKV. Following challenge with ZIKV, SCV-ZIKA/CHIK-vaccinated animals showed significant reductions in viremias compared with animals that had received a control SCV vaccine. Two SCV vaccinations also generated neutralising and IgG ELISA antibody responses to vaccinia virus. These results demonstrate effective induction of immunity in non-human primates by a recombinant SCV vaccine and illustrates the utility of SCV as a multi-disease vaccine platform capable of delivering multiple large immunogens.

Comparative Pathogenesis of Asian and African-Lineage Zika Virus in Indian Rhesus Macaque's and Development of a Non-Human Primate Model Suitable for the Evaluation of New Drugs and Vaccines.

The establishment of a well characterized non-human primate model of Zika virus (ZIKV) infection is critical for the development of medical interventions. In this study, challenging Indian rhesus macaques (IRMs) with ZIKV strains of the Asian lineage resulted in dose-dependent peak viral loads between days 2 and 5 post infection and a robust immune response which protected the animals from homologous and heterologous re-challenge. In contrast, viremia in IRMs challenged with an African lineage strain was below the assay’s lower limit of quantitation, and the immune response was insufficient to protect from re-challenge. These results corroborate previous observations but are contrary to reports using other African strains, obviating the need for additional studies to elucidate the variables contributing to the disparities. Nonetheless, the utility of an Asian lineage ZIKV IRM model for countermeasure development was verified by vaccinating animals with a formalin inactivated reference vaccine and demonstrating sterilizing immunity against a subsequent subcutaneous challenge.

Synergistic antiviral activity of Sofosbuvir and type-I interferons (α and ß) against Zika virus.

Zika virus (ZIKV) is transmitted by mosquitoes and causes Dengue-like illness, neurological symptoms such as Guillain-Barré Syndrome and microcephaly in children born to infected pregnant mothers. Recently, the World Health Organization (WHO) declared ZIKV infection as a Global Health Emergency. However, there are no known prophylactic or therapeutic measures against this virus. As a proof of concept toward combination therapeutic strategy against ZIKV, combinations of host-targeted (Interferon-α and Interferon-ß) and direct acting (Sofosbuvir) antivirals were evaluated in a hepatic cell line (Huh7) using a Cytoprotection (CP) assay. The combination of these antivirals resulted in synergistic inhibition of ZIKV infection in the in vitro CP assay. Additional testing in a ZIKV yield assay demonstrated that combination treatment of these antivirals conferred >2-log reduction in the release of viral RNA. Measurement of ZIKV proteins in the cells infected with multiple ZIKV strains isolated from different geographical regions (Americas, Asia, and Africa) using an immunofluorescence assay confirmed the effective antiviral activity of this combination against ZIKV. These results demonstrate the in vitro proof of concept (POC) for using a combination approach utilizing the strengths of both virus and host-targeted antivirals. These results suggest the effectiveness of the combination strategy in combating ZIKV, in the in vitro systems. Further evaluation of such combination therapies in vivo might provide an impetus for the development of effective ZIKV therapeutic strategies.

A sensitive virus yield assay for evaluation of Antivirals against Zika Virus.

Despite the rapid spread of Zika virus (ZIKV) infection and associated neurological complications in the America's, prophylactic or therapeutic countermeasures are not currently available. This is mostly due to the fact that until recently there was no presumed need for medical intervention since there was no association between ZIKV infection and significant human morbidity. Consequently, there are currently no tools due mostly to the lack of sensitive cell based assays amenable for identification of ZIKV inhibitors. To address this unmet need we have developed a cell based virus yield assay suitable for testing antivirals against Zika virus. Using bioinformatics, several isolates of ZIKV from the Americas, Africa, and Asia were analyzed for sequence similarity. The alignment data were then used to design primers targeting a ZIKV genomic region that was highly conserved among all the ZIKV isolates. Subsequently, primers were used in a sensitive, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay to detect ZIKV RNA. The qRT-PCR assay was found to be highly sensitive (lower limit of detection between-10-100 copies) and reproducible. Evaluation of the primers and probes used for ZIKV against another flavivirus (Dengue virus) demonstrated specificity of detection. To evaluate potential of qRT-PCR assay as an antiviral screening tool against ZIKV, Vero cells pretreated with Type I Interferons (IFN α) were infected with virus, followed by measurement of ZIKV RNA found in the cell culture supernatants using qRT-PCR assay. Dose-dependent antiviral activity of Type I Interferons and mycophenolic acid (MPA) against Zika virus in this cell culture system was confirmed using qRT-PCR. Due to reproducible assay performance, qPCR associated higher sensitivity and short duration of the assay time, this novel cell based assay will be very useful for confirming the activity of antivirals against ZIKV.

Development of a Zika Virus Infection Model in Cynomolgus Macaques.

Limited availability of Indian rhesus macaques (IRM) is a bottleneck to study Zika virus (ZIKV) pathogenesis and evaluation of appropriate control measures in non-human primates. To address these issues, we report here the Mauritian cynomolgus macaque (MCM) model for ZIKV infection. In brief, six MCMs (seronegative for Dengue and ZIKV) were subdivided into three cohorts with a male and female each and challenged with different doses of Asian [PRVABC59 (Puerto Rico) or FSS13025 (Cambodia)] or African (IBH30656) lineage ZIKV isolates. Clinical signs were monitored; and biological fluids (serum, saliva, and urine) and tissues (testes and brain) were assessed for viral load by quantitative reverse transcription polymerase chain reaction and neutralizing antibodies (Nab) by 50% Plaque Reduction Neutralization Test (PRNT50) at various times post-infection (p.i). PRVABC59 induced viremia detectable up to day 10, with peak viral load at 2-3 days p.i. An intermittent viremia spike was observed on day 30 with titers reaching 2.5 × 103 genomes/mL. Moderate viral load was observed in testes, urine and saliva. In contrast, FSS13025 induced viremia lasting only up to 6 days and detectable viral loads in testes but not in urine and saliva. Recurrent viremia was detected but at lower titers compare to PRVABC59. Challenge with either PRVABC59 or FSS13025 resulted in 100% seroconversion; with mean PRNT50 titers ranging from 597 to 5179. IBH30656 failed to establish infection in MCM suggesting that MCM are susceptible to infection with ZIKV isolates of the Asian lineage but not from Africa. Due to the similarity of biphasic viremia and Nab responses between MCM and IRM models, MCM could be a suitable alternative for evaluation of ZIKV vaccine and therapeutic candidates.

Impact of minimal tumor burden on antibody response to vaccination.

Four randomized phase III trials conducted recently in melanoma patients in the adjuvant setting have been based in part on the correlation between antibody responses in immunized patients and improved survival. Each of these randomized trials demonstrated no clinical benefit, although again there was a significant correlation between antibody response after vaccination and disease free and overall survival. To better understand this paradox, we established a surgical adjuvant model targeting GD2 ganglioside on EL4 lymphoma cells injected into the foot pad followed by amputation at variable intervals. Our findings are (1) comparable strong therapeutic benefit resulted from treatment of mice after amputation with a GD2-KLH conjugate vaccine or with anti-GD2 monoclonal antibody 3F8. (2) The strongest correlation was between antibody induction in response to vaccination and prolonged survival. (3) Antibody titers in response to vaccination in tumor challenged mice as compared to unchallenged mice were far lower despite the absence of detectable recurrences at the time. (4) The half life of administered 3F8 monoclonal antibody (but not control antibody) in challenged mice administered was significantly shorter than the half life of 3F8 antibody in unchallenged controls. The correlation between vaccine-induced antibody titers and prolonged survival may reflect, at least in part, increased tumor burden in antibody-negative mice. Absorption of vaccine-induced antibodies by increased, although not detected tumor burden may also explain the correlation between vaccine-induced antibody titers and survival in the adjuvant clinical trials described above.

Preparation and evaluation of unimolecular pentavalent and hexavalent antigenic constructs targeting prostate and breast cancer: a synthetic route to anticancer vaccine candidates.

Several novel, fully synthetic, carbohydrate-based antitumor vaccines have been assembled. Each construct consists of multiple cancer-related antigens displayed on a single polypeptide backbone. Recent advances in synthetic methodology have allowed for the incorporation of a complex oligosaccharide terminating in a sialic acid residue (i.e., GM2) as one of the carbohydrate antigens. Details of the vaccine synthesis as well as the results of preliminary immunological investigations are described herein.

Identification of DHBcAg as a potent carrier protein comparable to KLH for augmenting MUC1 antigenicity.

MUC1 is expressed at the cell surface of epithelial cancers. We have shown previously that MUC1 conjugated to keyhole limpet hemocyanin (KLH) plus the saponin immunological adjuvant QS-21 induces consistent high titer IgM and IgG antibodies in patients after treatment of their primary or metastatic cancers. KLH however is poorly soluble and heterogeneous making it difficult to work with, and we hypothesize that changing carrier proteins mid-way through a vaccination schedule would further increase antibody titers. Consequently, there is need for an alternative potent carrier protein. Duck Hepatitis B core antigen (DHBcAg) has a molecular weight of approximately 25kDa and is easily purified as a single band, but it self aggregates into particles of approximately 6.4x10(6)Da. Consequently, it is highly immunogenic, easy to work with and amenable to chemical and genetic conjugation to antigens such as MUC1. We compare here in mice the immunogenicity of MUC1 chemically conjugated to KLH or DHBcAg and MUC1-DHBcAg recombinant protein after an initial series of three vaccinations and then after an additional series of three vaccinations with the same or opposite carrier, all mixed with the saponin immunological adjuvant GPI-0100. High titer IgG antibodies were observed in all groups after the initial three vaccinations: MUC1-DHBcAg median ELISA titer 1/51200, RecMUC1-DHBcAg 1/25600 and MUC1-KLH 1/12800. This increased to 1/6553600 after the second set of three immunizations when the carrier remained the same in all three groups, but titers were significantly lower when the carriers were changed for the final three immunizations. These data demonstrate that DHBcAg is an excellent carrier protein and that changing carrier proteins does not further augment immunogenicity.

A preclinical study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancers.

Previously using a series of monovalent vaccines, we demonstrated that the optimal method for inducing an antibody response against cancer cell-surface antigens is covalent conjugation of the antigens to keyhole limpet hemocyanin (KLH) and the use of a saponin adjuvant. We have prepared a heptavalent-KLH conjugate vaccine containing the seven epithelial cancer antigens GM2, Globo H, Lewis(y), TF(c), Tn(c), STn(c), and glycosylated MUC1. In preparation for testing this vaccine in the clinic, we tested the impact on antibody induction of administering the individual conjugates plus adjuvant compared with a mixture of the seven conjugates plus adjuvant, and of several variables thought to augment immunogenicity. These include approaches for decreasing suppressor cell activity or increasing helper T-lymphocyte activity (low dose cyclophosphamide or anti-CTLA-4 MAb), different saponin adjuvants at various doses (QS-21 and GPI-0100), and different methods of formulation (lyophilization and use of polysorbate 80). We find that: (1). Immunization with the heptavalent-KLH conjugate plus GPI-0100 vaccine induces antibodies against the seven antigens of comparable titer to those induced by the individual-KLH conjugate vaccines, high titers of antibodies against Tn (median ELISA titer IgM/IgG 320/10240), STn (640/5120), TF (320/10240), MUC1 (80/20480), and globo H (640/40); while lower titers of antibodies against Lewis(y)()(160/0) and only occasional antibodies against GM2 are induced. (2). These antibodies reacted with the purified synthetic antigens by ELISA, and with naturally expressed antigens on the cancer cell surface by FACS. (3). None of the approaches for further altering the suppressor cell/helper T-cell balance nor changes to the standard formulation by lyophilization or use of polysorbate 80 had any impact on antibody titers. (4). An optimal dose of saponin adjuvant, QS-21 (50 microg) or GPI-0100 (1000 microg), is required for optimal antibody titers. This heptavalent vaccine is sufficiently optimized for testing in the clinic.

On the power of chemical synthesis: immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines.

Synthetic carbohydrate cancer vaccines have been shown to stimulate antibody-based immune responses in both preclinical and clinical settings. The antibodies have been observed to react in vitro with the corresponding natural carbohydrate antigens expressed on the surface of tumor cells, and are able to mediate complement-dependent and/or antibody-dependent cell-mediated cytotoxicity. Furthermore, these vaccines have proven to be safe when administered to cancer patients. Until recently, only monovalent antigen constructs had been prepared and evaluated. Advances in total synthesis have now enabled the preparation of multivalent vaccine constructs, which contain several different tumor-associated carbohydrate antigens. Such constructs could, in principle, serve as superior mimics of cell surface antigens and, hence, as potent cancer vaccines. Here we report preclinical ELISA-based evaluation of a TF-Le(y)-Tn bearing construct (compound 3) with native mucin glycopeptide architecture and a Globo-H-Le(y)-Tn glycopeptide (compound 4) with a nonnative structure. Mice were immunized with one or the other of these constructs as free glycopeptides or as keyhole lymphet hemocyanin conjugates. Either QS-21 or the related GPI-0100 were coadministered as adjuvants. Both keyhole lymphet hemocyanin conjugates induced IgM and IgG antibodies against each carbohydrate antigen, however, the mucin-based TF-Le(y)-Tn construct was shown to be less antigenic than the unnatural Globo-H-Le(y)-Tn construct. The adjuvants, although related, proved significantly different, in that GPI-0100 consistently induced higher titers of antibodies than QS-21. The presence of multiple glycans in these constructs did not appear to suppress the response against any of the constituent antigens. Compound 4, the more antigenic of the two constructs, was also examined by fluorescence activated cell sorter analysis. Significantly, from these studies it was shown that antibodies stimulated in response to compound 4 reacted with tumor cells known to selectively express the individual antigens. The results demonstrate that single vaccine constructs bearing several different carbohydrate antigens have the potential to stimulate a multifaceted immune response.