A J Paramyxovirus-vectored HIV vaccine induces humoral and cellular responses in mice.

Human immunodeficiency virus (HIV) infects and depletes CD4+ T-cells, resulting in Acquired Immunodeficiency Syndrome (AIDS) and death. Despite numerous clinical trials, there is no licensed HIV vaccine. The HIV envelope glycoprotein (env) is a major target for vaccine development, especially for the development of antibody-mediated protection. In this study, we used J paramyxovirus (JPV) as a viral vector to express HIV-env. We replaced the JPV small hydrophobic (SH) gene with HIV-env (rJPV-env). Intranasal rJPV-env immunization induced anti-HIV-gp120 IgG antibodies in mice. Furthermore, we examined the immunogenicity of homologous and heterologous prime/boost regimens with rJPV-env, parainfluenza virus 5 (rPIV5)-vectored HIV-env, and HIV-Gag-Env virus-like particles (VLPs). The rJPV-env/rPIV5-env heterologous prime/boost regimen induced the strongest humoral and cellular responses. Introducing a third dose of immunization, mice that received a viral-vectored prime had high levels of HIV-env-specific cellular responses, with group rJPV-env/rPIV5-env/VLP having the highest. Together, this work indicates that a heterologous combination of viral-vectored HIV-env vaccines and a HIV-Gag-Env VLP induces high levels of humoral and cellular responses against HIV in mice.

Combined intranasal and intramuscular parainfluenza 5-, simian adenovirus ChAdOx1- and poxvirus MVA-vectored vaccines induce synergistically HIV-1-specific T cells in the mucosa.

Introduction: The primary goal of this work is to broaden and enhance the options for induction of protective CD8+ T cells against HIV-1 and respiratory pathogens. Methods: We explored the advantages of the parainfluenza virus 5 (PIV5) vector for delivery of pathogen-derived transgenes alone and in combination with the in-human potent regimen of simian adenovirus ChAdOx1 prime-poxvirus MVA boost delivering bi-valent mosaic of HIV-1 conserved regions designated HIVconsvX. Results: We showed in BALB/c mice that the PIV5 vector expressing the HIVconsvX immunogens could be readily incorporated with the other two vaccine modalities into a single regimen and that for specific vector combinations, mucosal CD8+ T-cell induction was enhanced synergistically by a combination of the intranasal and intramuscular routes of administration. Discussion: Encouraging safety and immunogenicity data from phase 1 human trials of ChAdOx1- and MVA-vectored vaccines for HIV-1, and PIV5-vectored vaccines for SARS-CoV-2 and respiratory syncytial virus pave the way for combining these vectors for HIV-1 and other indications in humans.

Efficacy of Parainfluenza Virus 5 (PIV5)-vectored Intranasal COVID-19 Vaccine as a Single Dose Vaccine and as a Booster against SARS-CoV-2 Variants.

Immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has greatly reduced coronavirus disease 2019 (COVID-19)-related deaths and hospitalizations, but waning immunity and the emergence of variants capable of immune escape indicate the need for novel SARS-CoV-2 vaccines. An intranasal parainfluenza virus 5 (PIV5)-vectored COVID-19 vaccine CVXGA1 has been proven efficacious in animal models and blocks contact transmission of SARS-CoV-2 in ferrets. CVXGA1 vaccine is currently in human clinical trials in the United States. This work investigates the immunogenicity and efficacy of CVXGA1 and other PIV5-vectored vaccines expressing additional antigen SARS-CoV-2 nucleoprotein (N) or SARS-CoV-2 variant spike (S) proteins of beta, delta, gamma, and omicron variants against homologous and heterologous challenges in hamsters. A single intranasal dose of CVXGA1 induces neutralizing antibodies against SARS-CoV-2 WA1 (ancestral), delta variant, and omicron variant and protects against both homologous and heterologous virus challenges. Compared to mRNA COVID-19 vaccine, neutralizing antibody titers induced by CVXGA1 were well-maintained over time. When administered as a boost following two doses of a mRNA COVID-19 vaccine, PIV5-vectored vaccines expressing the S protein from WA1 (CVXGA1), delta, or omicron variants generate higher levels of cross-reactive neutralizing antibodies compared to three doses of a mRNA vaccine. In addition to the S protein, the N protein provides added protection as assessed by the highest body weight gain post-challenge infection. Our data indicates that PIV5-vectored COVID-19 vaccines, such as CVXGA1, can serve as booster vaccines against emerging variants.

Immunogenicity of Mumps Virus Genotype G Vaccine Candidates in Jeryl Lynn-Immunized Mice.

Mumps virus (MuV) causes a highly contagious human disease characterized by the enlargement of the parotid glands. In severe cases, mumps can lead to neurological complications such as aseptic meningitis and encephalitis. Vaccination with the attenuated Jeryl Lynn (JL) MuV vaccine has dramatically reduced the incidence of MuV infection. Recently, large outbreaks have occurred in vaccinated populations. The vaccine strain JL was generated from genotype A, while most current circulating strains belong to genotype G. In this study, we examined the immunogenicity and longevity of genotype G-based vaccines. We found that our recombinant genotype G-based vaccines provide robust neutralizing titers toward genotype G for up to 1 year in mice. In addition, we demonstrated that a third dose of a genotype G-based vaccine following two doses of JL immunization significantly increases neutralizing titers toward the genotype G strain. Our data suggest that after two doses of JL vaccination, which most people have received, a third dose of a genotype G-based vaccine can generate immunity against a genotype G strain. IMPORTANCE At present, most individuals have received two doses of the measles, mumps, and rubella (MMR) vaccine, which contains genotype A mumps vaccine. One hurdle in developing a new mumps vaccine against circulating genotype G virus is whether the new genotype G vaccine can generate immunity in humans that are immunized against genotype A virus. This work demonstrates that a novel genotype G-based vaccine can be effective in animals which received two doses of genotype A-based vaccine, suggesting that the lead genotype G vaccine may induce anti-G immunity in humans who have received two doses of the current vaccine, providing support for testing this vaccine in humans.

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication.

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family Pneumoviridae and genus Orthopneumovirus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. IMPORTANCE RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.

Evaluation of a New Viral Vaccine Vector in Mice and Rhesus Macaques: J Paramyxovirus Expressing Hemagglutinin of Influenza A Virus H5N1.

H5N1, an avian influenza virus, is known to circulate in many Asian countries, such as Bangladesh, China, Cambodia, Indonesia, and Vietnam. The current FDA-approved H5N1 vaccine has a moderate level of efficacy. A safe and effective vaccine is needed to prevent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in humans. Nonsegmented negative-sense single-stranded viruses (NNSVs) are widely used as a vector to develop vaccines for humans, animals, and poultry. NNSVs stably express foreign genes without integrating with the host genome. J paramyxovirus (JPV) is a nonsegmented negative-strand RNA virus and a member of the proposed genus Jeilongvirus in the family Paramyxoviridae. JPV-specific antibodies have been detected in rodents, bats, humans, and pigs, but the virus is not associated with disease in any species other than mice. JPV replicates in the respiratory tract of mice and efficiently expresses the virus-vectored foreign genes in tissue culture cells. In this work, we explored JPV as a vector for developing an H5N1 vaccine using intranasal delivery. We incorporated hemagglutinin (HA) of H5N1 into the JPV genome by replacing the small hydrophobic (SH) gene to generate a recombinant JPV expressing HA (rJPV-ΔSH-H5). A single intranasal administration of rJPV-ΔSH-H5 protected mice from a lethal HPAI H5N1 challenge. Intranasal vaccination of rJPV-ΔSH-H5 in rhesus macaques elicited antigen-specific humoral and cell-mediated immune responses. This work demonstrates that JPV is a promising vaccine vector. IMPORTANCE A highly pathogenic avian influenza (HPAI) H5N1 outbreak in Southeast Asia destroyed millions of birds. Transmission of H5N1 into humans resulted in deaths in many countries. In this work, we developed a novel H5N1 vaccine candidate using J paramyxovirus (JPV) as a vector and demonstrated that JPV is an efficacious vaccine vector in animals. Nonsegmented negative-sense single-stranded viruses (NNSVs) stably express foreign genes without integrating into the host genome. JPV, an NNSV, replicates efficiently in the respiratory tract and induces robust immune responses.

Protection of K18-hACE2 mice and ferrets against SARS-CoV-2 challenge by a single-dose mucosal immunization with a parainfluenza virus 5-based COVID-19 vaccine.

Transmission-blocking vaccines are urgently needed to reduce transmission of SARS-CoV 2, the cause of the COVID-19 pandemic. The upper respiratory tract is an initial site of SARS-CoV-2 infection and, for many individuals, remains the primary site of virus replication. An ideal COVID-19 vaccine should reduce upper respiratory tract virus replication and block transmission as well as protect against severe disease. Here, we optimized a vaccine candidate, parainfluenza virus 5 (PIV5) expressing the SARS-CoV-2 S protein (CVXGA1), and then demonstrated that a single-dose intranasal immunization with CVXGA1 protects against lethal infection of K18-hACE2 mice, a severe disease model. CVXGA1 immunization also prevented virus infection of ferrets and blocked contact transmission. This mucosal vaccine strategy inhibited SARS-CoV-2 replication in the upper respiratory tract, thus preventing disease progression to the lower respiratory tract. A PIV5-based mucosal vaccine provides a strategy to induce protective innate and cellular immune responses and reduce SARS-CoV-2 infection and transmission in populations.

Parainfluenza Virus 5 Priming Followed by SIV/HIV Virus-Like-Particle Boosting Induces Potent and Durable Immune Responses in Nonhuman Primates.

The search for a preventive vaccine against HIV infection remains an ongoing challenge, indicating the need for novel approaches. Parainfluenza virus 5 (PIV5) is a paramyxovirus replicating in the upper airways that is not associated with any animal or human pathology. In animal models, PIV5-vectored vaccines have shown protection against influenza, RSV, and other human pathogens. Here, we generated PIV5 vaccines expressing HIV envelope (Env) and SIV Gag and administered them intranasally to macaques, followed by boosting with virus-like particles (VLPs) containing trimeric HIV Env. Moreover, we compared the immune responses generated by PIV5-SHIV prime/VLPs boost regimen in naïve vs a control group in which pre-existing immunity to the PIV5 vector was established. We demonstrate for the first time that intranasal administration of PIV5-based HIV vaccines is safe, well-tolerated and immunogenic, and that boosting with adjuvanted trimeric Env VLPs enhances humoral and cellular immune responses. The PIV5 prime/VLPs boost regimen induced robust and durable systemic and mucosal Env-specific antibody titers with functional activities including ADCC and neutralization. This regimen also induced highly polyfunctional antigen-specific T cell responses. Importantly, we show that diminished responses due to PIV5 pre-existing immunity can be overcome in part with VLP protein boosts. Overall, these results establish that PIV5-based HIV vaccine candidates are promising and warrant further investigation including moving on to primate challenge studies.

Evaluation of the Pig-Tailed Macaque (Macaca nemestrina) as a Model of Human Staphylococcus aureus Nasal Carriage.

Staphylococcus aureus nasal carriage is a common condition affecting both healthy and immunocompromised populations and provides a reservoir for dissemination of potentially infectious strains by casual contact. The factors regulating the onset and duration of nasal S. aureus colonization are mostly unknown, and a human-relevant animal model is needed. Here, we screened 17 pig-tailed macaques (Macaca nemestrina) for S. aureus carriage, and 14 of 17 animals tested positive in the nose at one or both screening sessions (8 weeks apart), while the other 3 animals were negative in the nose but positive in the pharynx at least once. As in humans, S. aureus colonization was densest in the nose, and treatment of the nostrils with mupirocin ointment effectively cleared the nostrils and 6 extranasal body sites. Experimental nasal S. aureus colonization was established with 104 CFU/nostril, and both autologous and nonautologous strains survived over 40 days without any apparent adverse effects. A human nasal S. aureus isolate (strain D579, sequence type 398) was carried in 4 of 6 animals for over 3 weeks. Nostrils that did eradicate experimentally applied S. aureus exhibited neutrophilic innate immunity marked by elevated nasal interleukin-1ß (IL-1ß), IL-8, and monocyte chemotactic protein 1 levels and a 10-fold decreased IL-1 receptor antagonist/IL-1ß ratio within 7 days postinoculation, analogous to the human condition. Taken together, pig-tailed macaques represent a physiological model of human S. aureus nasal carriage that may be utilized for testing natural colonization and decolonization mechanisms as well as novel classes of anti-S. aureus therapeutics.

Cessation from Smoking Improves Innate Host Defense and Clearance of Experimentally Inoculated Nasal Staphylococcus aureus.

Staphylococcus aureus nasal carriage is transient in most humans and usually benign, but dissemination of S. aureus to extranasal sites causes the majority of clinical infections, and S. aureus is a major cause of serious infections in the United States. A better understanding of innate nasal decolonization mechanisms is urgently needed, as are relevant models for studying S. aureus clearance. Here, we screened a population of healthy smokers for nasal S. aureus carriage and compared the participants' abilities to clear experimentally applied nasal S. aureus before and after completion of a smoking cessation program. We determined that cigarette smoking increases the mean nasal S. aureus load (2.6 × 104 CFU/swab) compared to the load observed in healthy nonsmokers (1.7 × 103 CFU/swab) and might increase the rate of S. aureus nasal carriage in otherwise-healthy adults: 22 of 99 smokers carried S. aureus at the screening visit, while only 4 of 30 nonsmokers screened positive during the same time period. Only 6 of 19 experimental inoculation studies in active smokers resulted in S. aureus clearance within the month of follow-up, while in the cessation group, 6 of 9 subjects cleared nasal S. aureus and carriage duration averaged 21 ± 4 days. Smoking cessation associated with enhanced expression of S. aureus-associated interleukin-1ß (IL-1ß) and granulocyte colony-stimulating factor (G-CSF) in nasal fluids. Participants who failed to clear S. aureus exhibited a higher nasal S. aureus load and elevated nasal interleukin-1 receptor antagonist (IL-1RA) expression at the preexperiment study visits. We conclude that smokers exhibit higher S. aureus loads than nonsmokers and that innate immune pathways, including G-CSF expression and signaling through the IL-1 axis, are important mediators of nasal S. aureus clearance.