Novel immunomodulatory properties of adenosine analogs promote their antiviral activity against SARS-CoV-2.

The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.

Longitudinal Variations in Antibody Responses against SARS-CoV-2 Spike Epitopes upon Serial Vaccinations.

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacted healthcare, the workforce, and worldwide socioeconomics. Multi-dose mono- or bivalent mRNA vaccine regimens have shown high efficacy in protection against SARS-CoV-2 and its emerging variants with varying degrees of efficacy. Amino acid changes, primarily in the receptor-binding domain (RBD), result in selection for viral infectivity, disease severity, and immune evasion. Therefore, many studies have centered around neutralizing antibodies that target the RBD and their generation achieved through infection or vaccination. Here, we conducted a unique longitudinal study, analyzing the effects of a three-dose mRNA vaccine regimen exclusively using the monovalent BNT162b2 (Pfizer/BioNTech) vaccine, systematically administered to nine previously uninfected (naïve) individuals. We compare changes in humoral antibody responses across the entire SARS-CoV-2 spike glycoprotein (S) using a high-throughput phage display technique (VirScan). Our data demonstrate that two doses of vaccination alone can achieve the broadest and highest magnitudes of anti-S response. Moreover, we present evidence of novel highly boosted non-RBD epitopes that strongly correlate with neutralization and recapitulate independent findings. These vaccine-boosted epitopes could facilitate multi-valent vaccine development and drug discovery.

Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia.

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4(+) T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD11b(+) macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina-specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

Dynamics of Serum-Neutralizing Antibody Responses in Vaccinees through Multiple Doses of the BNT162b2 Vaccine.

SARS-CoV-2 mRNA vaccines are administered as effective prophylactic measures for reducing virus transmission rates and disease severity. To enhance the durability of post-vaccination immunity and combat SARS-CoV-2 variants, boosters have been administered to two-dose vaccinees. However, long-term humoral responses following booster vaccination are not well characterized. A 16-member cohort of healthy SARS-CoV-2 naïve participants were enrolled in this study during a three-dose BNT162b2 vaccine series. Serum samples were collected from vaccinees over 420 days and screened for antigen (Ag)-specific antibody titers, IgG subclass distribution, and neutralizing antibody (nAb) responses. Vaccine boosting restored peak Ag-specific titers with sustained α-RBD IgG and IgA antibody responses when measured at six months post-boost. RBD- and spike-specific IgG4 antibody levels were markedly elevated in three-dose but not two-dose immune sera. Although strong neutralization responses were detected in two- and three-dose vaccine sera, these rapidly decayed to pre-immune levels by four and six months, respectively. While boosters enhanced serum IgG Ab reactivity and nAb responses against variant strains, all variants tested showed resistance to two- and three-dose immune sera. Our data reflect the poor durability of vaccine-induced nAb responses which are a strong predictor of protection from symptomatic SARS-CoV-2 infection. The induction of IgG4-switched humoral responses may permit extended viral persistence via the downregulation of Fc-mediated effector functions.

Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples.

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. Here we report a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. We adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

Intranasal boosting with MVA encoding secreted mycobacterial proteins Ag85A and ESAT-6 generates strong pulmonary immune responses and protection against M. tuberculosis in mice given BCG as neonates.

Bacille-Calmette-Guerin (BCG) has variable efficacy as an adult tuberculosis (TB) vaccine but can reduce the incidence and severity of TB infection in humans. We have engineered modified vaccinia Ankara (MVA) strain vaccine constructs to express the secreted mycobacterial proteins Ag85A and ESAT-6 (MVA-AE) and evaluated their immunogenicity and protective efficacy as mucosal booster vaccines for BCG given subcutaneously in early life. Intranasal delivery of MVA-AE to young adult mice induced CD4+ and CD8+ T cell responses to both Ag85A and ESAT-6 in lung mucosae. These responses were markedly enhanced in mice that had been primed neonatally with BCG prior to intranasal MVA-AE immunization (BCG/MVA-AE), as evidenced by numbers of pulmonary Ag85A-, ESAT-6-, and PPD-specific CD4+ and CD8+ T cells and by their capacity to secrete multiple antimicrobial factors, including IFNγ, IL-2 and IL-17. Moreover, MVA-AE boosting generated multifunctional lung CD4+ T cells responding to ESAT-6, which were not, as expected, detected in control mice given BCG, and elevated Ag85A-specific circulating antibody responses. After aerosol challenge with M. tuberculosis H37Rv (Mtb), the BCG/MVA-AE group had significantly reduced mycobacterial burden in the lungs, compared with either BCG primed mice boosted with control MVA or mice given only BCG. These data indicate that intranasal delivery of MVA-AE can boost BCG-induced Th1 and Th17-based immunity locally in the lungs and improve the protective efficacy of neonatally-administered BCG against M. tuberculosis infection.

CD4+ T cell-independent DNA vaccination against opportunistic infections.

Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modified DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

B cell and antibody responses in mice induced by a putative cell surface peptidase of Pneumocystis murina protect against experimental infection.

RATIONALE: Pneumocystis pneumonia is a major cause of morbidity and mortality in HIV-infected subjects, cancer patients undergoing chemotherapy and solid organ transplant recipients. No vaccine is currently available. By chemical labeling coupled with proteomic approach, we have identified a putative surface protein (SPD1, Broad Institute gene accession number PNEG_01848) derived from single suspended P. murina cysts. SPD1 was expressed in an insect cell line and tested for vaccine development. METHODS: Mice were immunized with SPD1 plus adjuvant MF-59 by subcutaneous injection. Three weeks after the last immunization, CD4+ cells were depleted with anti-CD4 antibody GK1.5. The mice were then challenged with 2×105Pneumocystis organisms. Mice were sacrificed at 4 and 6weeks after PC challenge. Spleen/lung cells and serum were harvested. B cells and memory B cells were assessed via flow cytometry. Specific Pneumocystis IgG antibody was measured by ELISA before and after challenge. Infection burden was measured as real-time PCR for P. murina rRNA. RESULTS: Normal mice infected with Pneumocystis mounted a serum IgG antibody response to SPD1. Serum from rhesus macaques exposed to Pneumocystis showed a similar serum IgG response to purified SPD1. SPD1 immunization increased B cell and memory B cell absolute cell counts in CD4-depleted Balb/c mice post Pneumocystis challenge in spleen and lung. Immunization with SPD1 significantly increased specific Pneumocystis IgG antibody production before and after challenge. Mice immunized with SPD1 showed significantly decreased P. murina copy number compared with mice that did not receive SPD1 at 6weeks after challenge. CONCLUSION: Immunization with SPD1 provides protective efficacy against P. murina infection. SPD1 protection against Pneumocystis challenge is associated with enhanced memory B cell production and higher anti-Pneumocystis IgG antibody production. SPD1 is a potential vaccine candidate to prevent or treat pulmonary infection with Pneumocystis.

Flagellin Encoded in Gene-Based Vector Vaccines Is a Route-Dependent Immune Adjuvant.

Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route.

Gene-based neonatal immune priming potentiates a mucosal adenoviral vaccine encoding mycobacterial Ag85B.

Tuberculosis remains a major public health hazard worldwide, with neonates and young infants potentially more susceptible to infection than adults. BCG, the only vaccine currently available, provides some protection against tuberculous meningitis in children but variable efficacy in adults, and is not safe to use in immune compromised individuals. A safe and effective vaccine that could be given early in life, and that could also potentiate subsequent booster immunization, would represent a significant advance. To test this proposition, we have generated gene-based vaccine vectors expressing Ag85B from Mycobacterium tuberculosis (Mtb) and designed experiments to test their immunogenicity and protective efficacy particularly when given in heterologous prime-boost combination, with the initial DNA vaccine component given soon after birth. Intradermal delivery of DNA vaccines elicited Th1-based immune responses against Ag85B in neonatal mice but did not protect them from subsequent aerosol challenge with virulent Mtb H37Rv. Recombinant adenovirus vectors encoding Ag85B, given via the intranasal route at six weeks of age, generated moderate immune responses and were poorly protective. However, neonatal DNA priming following by mucosal boosting with recombinant adenovirus generated strong immune responses, as evidenced by strong Ag85B-specific CD4+ and CD8+ T cell responses, both in the lung-associated lymph nodes and the spleen, by the quality of these responding cells (assessed by their capacity to secrete multiple antimicrobial factors), and by improved protection, as indicated by reduced bacterial burden in the lungs following pulmonary TB challenge. These results suggest that neonatal immunization with gene-based vaccines may create a favorable immunological environment that potentiates the pulmonary mucosal boosting effects of a subsequent heterologous vector vaccine encoding the same antigen. Our data indicate that immunization early in life with mycobacterial antigens in an appropriate vaccine setting can prime for protective immunity against Mtb.

The efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin-4.

Improved vaccines and therapies for virulent poxvirus infection are required, particularly in the light of recent threats of bioterrorism. Cidofovir (HPMPC) is an acyclic nucleoside analog with proven efficacy against poxviruses. Here, we evaluated HPMPC in mice given a recombinant ectromelia virus (ECTV) encoding interleukin-4 (ECTV-IL-4) that is highly immune suppressive. Mousepox-sensitive BALB/c mice given HPMPC for five consecutive days after infection were protected against the lethal effects of a control ECTV recombinant, although they suffered a chronic form of mousepox disease. High doses of the drug resulted in a milder localized disease. In contrast, HPMPC failed to protect mousepox-resistant C57BL/6 mice against ECTV-IL-4, although its lethal effects were delayed by five daily doses of 20 mg/kg or a single dose of 100 mg/kg. Higher daily doses further delayed mortality, although the majority of animals eventually succumbed to infection. It appears that HPMPC inhibited ECTV-IL-4 replication without clearance, with the virus having a lethal effect when the drug was removed. Resistance of ECTV-IL-4 to HPMPC treatment may relate to the virus's ability to inhibit antiviral cell-mediated immunity. Interestingly, ECTV-IL-4-mediated immune suppression was not accompanied by a reduction in systemic IFN-gamma expression, suggestive of an alternative or highly localized suppressive mechanism.

DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection.

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.

Prime-boost approaches to tuberculosis vaccine development.

Four individuals die from active TB disease each minute, while at least 2 billion are latently infected and at risk for disease reactivation. BCG, the only licensed TB vaccine, is effective in preventing childhood forms of TB; however its poor efficacy in adults, emerging drug-resistant TB strains and tedious chemotherapy regimes, warrant the development of novel prophylactic measures. Designing safe and effective vaccines against TB will require novel approaches on several levels, including the administration of rationally selected mycobacterial antigens in efficient delivery vehicles via optimal immunization routes. Given the primary site of disease manifestation in the lungs, development of mucosal immunization strategies to generate protective immune responses both locally, and in the circulation, may be important for effective TB prophylaxis. This review focuses on prime-boost immunization strategies currently under investigation and highlights the potential of mucosal delivery and rational vaccine design based on systems biology.

Efficient generation and rapid isolation via stoplight recombination of Herpes simplex viruses expressing model antigenic and immunological epitopes.

Generation and isolation of recombinant herpesviruses by traditional homologous recombination methods can be a tedious, time-consuming process. Therefore, a novel stoplight recombination selection method was developed that facilitated rapid identification and purification of recombinant viruses expressing fusions of immunological epitopes with EGFP. This "traffic-light" approach provided a visual indication of the presence and purity of recombinant HSV-1 isolates by producing three identifying signals: (1) red fluorescence indicates non-recombinant viruses that should be avoided; (2) yellow fluorescence indicates cells co-infected with non-recombinant and recombinant viruses that are chosen with caution; (3) green fluorescence indicates pure recombinant isolates and to proceed with preparation of viral stocks. Adaptability of this system was demonstrated by creating three recombinant viruses that expressed model immunological epitopes. Diagnostic PCR established that the fluorescent stoplight indicators were effective at differentiating between the presence of background virus contamination and pure recombinant viruses specifying immunological epitopes. This enabled isolation of pure recombinant viral stocks that exhibited wildtype-like viral replication and cell-to-cell spread following three rounds of plaque purification. Expression of specific immunological epitopes was confirmed by western analysis, and the utility of these viruses for examining host immune responses to HSV-1 was determined by a functional T cell assay.

CD40 ligand enhances immunogenicity of vector-based vaccines in immunocompetent and CD4+ T cell deficient individuals.

Impairment of host immunity, particularly CD4+ T cell deficiency, presents significant complications for vaccine immunogenicity and efficacy. CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor superfamily (TNFSF), is an important co-stimulatory molecule and, through interactions with its cognate receptor CD40, plays a pivotal role in the generation of host immune responses. Exploitation of CD40L and its receptor CD40 could provide a means to enhance and potentially restore protective immune responses in CD4+ T cell deficiency. To investigate the potential adjuvanticity of CD40L, we constructed recombinant plasmid DNA and adenoviral (Ad) vaccine vectors expressing murine CD40L and the mycobacterial protein antigen 85B (Ag85B). Co-immunization of mice with CD40L and Ag85B by intranasal or intramuscular prime-boosting led to route-dependent enhancement of the magnitude of vaccine-induced circulating and lung mucosal CD4+ and CD8+ T cell responses in both normal (CD4-replete) and CD4+ T cell deficient animals, including polyfunctional T cell responses. The presence of CD40L alone was insufficient to enhance or restore CD4+ T cell responses in CD4-ablated animals; however, in partially depleted animals, co-immunization with Ag85B and CD40L was capable of eliciting enhanced T cell responses, similar to those observed in normal animals, when compared to those given vaccine antigen alone. In summary, these findings show that CD40L has the capacity to enhance the magnitude of vaccine-induced polyfunctional T cell responses in CD4+ T cell deficient mice, and warrants further study as an adjuvant for immunization against opportunistic pathogens in individuals with CD4+ T cell deficiency.

A comparative analysis of HIV-specific mucosal/systemic T cell immunity and avidity following rDNA/rFPV and poxvirus-poxvirus prime boost immunisations.

In this study we have firstly compared a range of recombinant DNA poxvirus prime-boost immunisation strategies and shown that combined intramuscular (i.m.) 2× DNA-HIV/intranasal (i.n.) 2× FPV-HIV prime-boost immunisation can generate high-level of HIV-specific systemic (spleen) and mucosal (genito-rectal nodes, vaginal tissues and lung tissues) T cell responses and HIV-1 p24 Gag-specific serum IgG1, IgG2a and mucosal IgG, SIgA responses in vaginal secretions in BALB/c mice. Data indicate that following rDNA priming, two rFPV booster immunisations were necessary to generate good antibody and mucosal T cell immunity. This data also revealed that mucosal uptake of recombinant fowl pox (rFPV) was far superior to plasmid DNA. To further evaluate CD8+ T cell immunity, i.m. 2× DNA-HIV/i.n. 1× FPV-HIV immunisation strategy was directly compared with single shot poxvirus/poxvirus, i.n. FPV-HIV/i.m. VV-HIV immunisation. Results indicate that the latter strategy was able to generate strong sustained HIV-specific CD8+ T cells with higher avidity, broader cytokine/chemokine profiles and better protection following influenza-K(d)Gag(197-205) challenge compared to rDNA poxvirus prime-boost strategy. Our findings further substantiate the importance of vector selection/combination, order and route of delivery when designing effective vaccines for HIV-1.

Single-shot immunization with recombinant adenovirus encoding vaccinia virus glycoprotein A27L is protective against a virulent respiratory poxvirus infection.

Significant safety issues have emerged concerning the general use of DRYVAX vaccine. Vaccination with replication-defective recombinant adenovirus (rAd) vaccines may offer a safer and effective alternative to live vaccinia virus (VV) vaccination. Six individual poxvirus glycoproteins: A33R, A34R, A36R, B5R, A27L or L1R that are normally expressed on the surface of infectious vaccinia virus were encoded in rAd vaccines and tested in mice in this study. A single-shot intramuscular injection of rAd encoding A27L protected mice against a lethal intranasal challenge with VV at 4 weeks post-vaccination. By 10 weeks post-vaccination, a significant decrease in post-challenge morbidity was observed that correlated with potent neutralizing antibody responses and the emergence of specific polyfunctional T cell responses. The immunogenicity and protective efficacy of rAd-A27L immunization persisted for at least 35 weeks post-vaccination. This study is the first demonstration that a single-shot subunit vaccine encoding a poxvirus protein confers protection against the mortality and morbidity associated with poxvirus infection.

Differential effects of the type I interferons alpha4, beta, and epsilon on antiviral activity and vaccine efficacy.

The type I IFNs exert a range of activities that include antiviral, antiproliferative, and immunomodulatory effects. To study this further, we have constructed recombinant vaccinia viruses expressing HIV or hemagglutinin (HA) Ags along with murine type I IFNs, IFN-alpha(4) (HA-VV-IFN-alpha(4)), IFN-beta (HA-VV-IFN-beta), or IFN-epsilon (HIV-VV-IFN-epsilon), a recently discovered member of this family. Our aims were to characterize IFN-epsilon functionality as a type I IFN and also to study the biological properties of these factors toward the development of safer and more effective vector-based vaccines. HIV-VV-IFN-epsilon and HA-VV-IFN-beta grew to lower titers than did their parental controls in murine cell lines. In vivo, however, HIV-VV-IFN-epsilon growth was not attenuated, while IFN-beta demonstrated potent local antiviral activity with no replication of HA-VV-IFN-beta detected. Flow cytofluorometric analysis of B lymphocytes incubated with virally encoded IFN-epsilon showed up-regulation of activation markers CD69 and CD86, while RT-PCR of IFN-epsilon-treated cells revealed that gene expression levels of antiviral proteins were elevated, indicating the induction of an antiviral state. The use of these constructs in a poxvirus prime-boost immunization regime led to robust humoral and cellular immune responses against the encoded Ags, despite the lack of replication in the case of HA-VV-IFN-beta. Thus, coexpression of these factors may be beneficial in the design of safer vector-based vaccines. Our data also indicate that while IFN-epsilon exhibits certain biological traits similar to other type I IFNs, it may also have a specific role in mucosal immune regulation that is quite distinct.

Evaluation of fowlpox-vaccinia virus prime-boost vaccine strategies for high-level mucosal and systemic immunity against HIV-1.

We have tested the efficacy of recombinant fowl pox (rFPV) and recombinant vaccinia virus (rVV) encoding antigens of AE clade HIV-1 in a prime-boost strategy, using both systemic and mucosal delivery routes. Of the various vaccine routes tested, intranasal/intramuscular (i.n./i.m.) AE FPV/AE VV prime-boosting generated the highest mucosal and systemic T cell responses. Peak mucosal T cell responses occurred as early as 3 days post-boost vaccination. In contrast only low systemic responses were observed at this time with the peak response occurring at day 7. Current data also revealed that, due to better uptake of the rFPV, intranasal viral priming was much more effective than intranasal rDNA priming tested previously. The i.m./i.m. prime-boost delivery also generated strong systemic but poor mucosal responses to Gag peptides. Interestingly, the oral administration of AE FPV followed by i.m. AE VV delivery elicited strong systemic responses to sub-dominant Pol 1 peptides that were absent in mice that received vaccine by other routes. Moreover, priming with AE FPV co-expressing cytokine IL-12 significantly enhanced the T cell responses to target antigens, whilst co-expression of IFNgamma decreased these responses. The results also indicated that the route of inoculation and the vaccine vector combination could radically influence not only the magnitude but also the antigen specificity of the immune response generated. Further, in contrast to the generally protracted HIV rDNA/rFPV multiple delivery prime-boosting, this single rFPV prime and rVV boost approach was more flexible and generated excellent mucosal and systemic immune responses to HIV vaccine antigens.