Recombinant Poxvirus Research Articles

Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

BackgroundThere is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes.MethodsThree Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl β-galactosidase assay with primary isolates of HIV-1.ResultsThis study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the antibodies did not neutralise primary isolates of HIV-1 or the V3-sensitive isolate SF162 using the TZM-bl β-galactosidase assay.ConclusionsMVA and FP9 are ideal replication-deficient viral vectors for HIV-1 vaccines due to their excellent safety profile for use in humans. This study shows this novel prime-boost-boost regimen was poorly immunogenic in Chinese cynomolgus macaques.

A Poxvirus Vaccine Is Safe, Induces T-Cell Responses, and Decreases Viral Load in Patients With Chronic Hepatitis C

Therapy for chronic hepatitis C (CHC) has limited efficacy, adverse effects, and high costs. Cohort and vaccine-based preclinical studies have indicated the importance of T-cell-based immunity in controlling viral infection. TG4040 is a recombinant poxvirus vaccine that expresses the hepatitis C virus (HCV) proteins NS3, NS4, and NS5B. We performed a phase I clinical trial to assess the safety, immunogenicity, and early antiviral efficacy of TG4040 in patients with CHC. In an open-label, dose-escalating study, patients with mild CHC (genotype 1) were assigned to 3 groups of 3 patients each; they received subcutaneous injections of 10⁶, 10⁷, or 10⁸ plaque-forming units of TG4040 on study days 1, 8, and 15. Six additional patients were given the highest dose of vaccine (10⁸ plaque-forming units). Patients were followed for 6 months after the last injection. T-cell-based and antibody responses and levels of HCV RNA were measured. All 3 doses of TG4040 were well tolerated, without serious adverse events. Vaccine-induced HCV-specific cellular immune responses were observed in 5 of the 15 patients (33%). A transient decrease in circulating levels of HCV RNA, from -0.52 log₁₀ to -1.24 log₁₀, was observed in 8 patients; in 5 patients, the lowest level of HCV RNA was observed on day 37, after the first injection. The most pronounced decrease in viral load occurred in 2 patients, who also had marked vaccine-induced T-cell responses. In patients with CHC, the viral-vector-based vaccine TG4040 had a good safety profile, induced HCV-specific cellular immune responses, and reduced viral load. This vaccine should be investigated in further clinical studies, in combination with standard of care.

Intraprostatic PSA-TRICOM vaccine administration in patients with locally recurrent prostate cancer.

2530 Background: PSA-TRICOM is a cancer vaccine composed of recombinant poxviruses with transgenes for PSA and 3 T cell costimulatory molecules (TRICOM). In a multicenter randomized vector controlled phase II study median overall survival improved by 8.5 months in men with metastatic CRPC. Preclinical studies show intratumoral administration of TRICOM vaccines may improve anti-tumor effect. This study intended to show safety of intraprostatic (i.p.) vaccine administration. 2° goals were immunologic and tumor response. Methods: 21 pts with locally recurrent prostate cancer after radiation were enrolled into 5 cohorts and received initial vaccination with s.c. recombinant vaccinia (rV)-PSA-TRICOM and booster i.p. recombinant fowlpox (rF)-PSA-TRICOM. Cohorts 3-5 also received i.p. rF-GM-CSF. Cohort 5 received concurrent s.c. and i.p. boosters. Priming was given on day 1. Boosters were given on days 29, 57, and 85. Pts had pre- and post-treatment prostate biopsies. Tumor infiltrate was evaluated when adequate tissue was available. PBMCs were analyzed by ELISPOT for response to PSA and 3-color flow cytometry to characterize regulatory T cells (Treg) or effector T cells. Treg suppression function was assayed by suppression of the CD4+ effector T cell proliferation. Results: Only 1 grade 3 toxicity occurred, a transient fever. Most common grade 2 adverse events for all doses were fever (14.5%) and injection-site reactions (21%). 18 of 21 pts had stable or improved PSA on study. 16 of 21 pts had stable or improved PSA doubling time. Post-vaccination, 4 of 8 evaluable pts had immune response by ELISPOT and 11 of 15 pts had decreased (n=8) or stable (n=3) Treg function. A paired t-test of 13 biopsies pre- and post-vaccination showed increases in tumor immunologic infiltrates. CD3+ cells increased from 12.3 → 21.3/high power field (hpf) (p=0.0079). CD4+ cells increased from 1.3 →13.1/hpf (p=0.0002). CD8+ cells rose from 6.4 →14/hpf (p=0.0002). Conclusions: I.p. administration of PSA-TRICOM is safe, feasible and can generate a substantial immune response. Improved PSA kinetics and intense post-vaccination immune infiltrates were seen in the majority of pts. Trials of i.p. PSA-TRICOM with clinical endpoints are warranted.

Vaccinia virus as a vaccine delivery system for marsupial wildlife

Vaccines based on recombinant poxviruses have proved successful in controlling diseases such as rabies and plague in wild eutherian mammals. They have also been trialled experimentally as delivery agents for fertility-control vaccines in rodents and foxes. In some countries, marsupial mammals represent a wildlife disease reservoir or a threat to conservation values but, as yet there has been no bespoke study of efficacy or immunogenicity of a poxvirus-based vaccine delivery system in a marsupial. Here, we report a study of the potential for vaccination using vaccinia virus in the Australian brushtail possum Trichosurus vulpecula, an introduced pest species in New Zealand. Parent-strain vaccinia virus (Lister) infected 8/8 possums following delivery of virus to the oral cavity and outer nares surfaces (oronasal immunisation), and persisted in the mucosal epithelium around the palatine tonsils for up to 2 weeks post-exposure. A recombinant vaccinia virus construct (VV399, which expresses the Eg95 antigen of the hydatid disease parasite Echinococcus granulosus) was shown to infect 10/15 possums after a single-dose oronasal delivery and to also persist. Both parent vaccinia virus and the VV399 construct virus induced peripheral blood lymphocyte reactivity against viral antigens in possums, first apparent at 4 weeks post-exposure and still detectable at 4 months post-exposure. Serum antibody reactivity to Eg95 was recorded in 7/8 possums which received a single dose of the VV399 construct and 7/7 animals which received triple-dose delivery, with titre end-points in the latter case exceeding 1/4000 dilution. This study demonstrates that vaccinia virus will readily infect possums via a delivery means used to deploy wildlife vaccines, and in doing is capable of generating immune reactivity against viral and heterologous antigens. This highlights the future potential of recombinant vaccinia virus as a vaccine delivery system in marsupial wildlife.

Characterization of the human endogenous retrovirus K Gag protein: identification of protease cleavage sites

BackgroundViral genomes of the human endogenous retrovirus K (HERV-K) family are integrated into the human chromosome and are transmitted vertically as Mendelian genes. Although viral particles are released by some transformed cells, they have never been shown to be infectious. In general, gammaretroviruses are produced as immature viral particles by accumulation of the Gag polyproteins at the plasma membrane, which subsequently bud from the cell surface. After release from the cell, Gag is further processed by proteolytic cleavage by the viral protease (PR), which results in morphologically mature particles with condensed cores. The HERV-K Gag polyprotein processing and function has not yet been precisely determined.ResultsWe generated a recombinant poxvirus, encoding the human endogenous retrovirus K consensus gag-pro-pol genes (MVA-HERV-Kcon) and obtained high levels of HERV-K Gag expression. The resulting retroviral particle assembled at the plasma membrane, as is typical for gammaretroviruses; and immature as well as mature retrovirus-like particles (VLPs) were observed around the infected cells. VLPs were purified, concentrated and separated by two-dimensional gel electrophoresis. The HERV-K Gag fragments were identified by mass spectroscopy and N-terminal sequencing which revealed that HERV-K Gag is processed into MA, a short spacer peptide, p15, CA and NC.ConclusionThe cleavage sites of HERV-K Gag were mapped and found to be highly conserved among HERV-K genomes. The consensus HERV-K gag gene used in this study is known to support viral, infectivity [1], and thus the cleavage sites that were mapped in this study for all the Gag components are relevant for HERV-K infectivity.

An Efficient Method for Generating Poxvirus Recombinants in the Absence of Selection

The use of selectable markers (ecogpt) and selection pressures to aid in detection of poxvirus (Vaccinia, VV) recombinants has been implicated in the unintended introduction of second site mutations. We have reinvestigated the use of the helper virus system described by Scheiflinger et al. [1] and adapted by Yao and Evans [2] which produces recombinants at a high frequency in the absence of any selection, at a rate of 6–100%. Our system uses fowlpox virus (FPV) as the infectious helper virus which in infected cells provides the enzymatic apparatus for transcription and replication of a purified, transfected VV genome and for recombination with a second transfected PCR generated DNA fragment. To optimize the system, a PCR DNA fragment was generated that contained poxvirus promoter driven gfp and lacZ genes inserted within the coding sequences of the viral thymidine kinase gene. This PCR fragment was co-transfected together with VV genomic DNA. Recombinant VV was identified by plaquing the mixture on cells non-permissive for FPV and selection of green fluorescent or LacZ positive recombinant vaccinia plaques. The system was optimized using FPV permissive cells (CEF) and non-permissive cells (A549, CV-1) for both the initial infection/transfection and the subsequent selection. Up to 70% of the progeny vaccinia virus contained the gfp/LacZ insertion. In order to test for the presence of FPV/VV intertypic recombinants or other unintended mutations, recombinant wtVV (RwtVV) was regenerated from the gfp/LacZ viruses and evaluated by RFLP analysis and pathogenesis in animals. While all RwtVVs were viable in cell culture, in many of the RwtVV isolates, RFLP differences were noted and while some recombinant viruses exhibited wild type behavior in mice, a wide range of virulence indicative of unintended changes suggests that mutants created by “rescue” systems require careful analysis particularly before use for in vivo studies employing animal models.

HIV vaccines: progress to date.

The quest for an effective and safe HIV-1 vaccine has been and still is the aspiration of many scientists and clinicians worldwide. Until recently, the hopes for an effective vaccine were thwarted by the disappointing results and early termination in September 2007 of the STEP study, which saw a subgroup of male vaccine recipients at an increased risk of HIV-1 infection, and the failure of earlier trials of vaccines based on recombinant envelope proteins to provide any level of protection. The results of the STEP study raised important questions in the field of HIV vaccines, including the use of recombinant adenovirus vectors as immunogens, the rationale for the development of T-cell-based vaccines and the development pathway for these vaccines, in terms of assessment of immunogenicity and the challenge models used. The study of neutralizing antibodies has demonstrated that the induction of high-titre, broadly neutralizing antibodies in the majority of recipients is likely to be highly problematic. However, the results of the RV144 Thai trial released in September 2009 have brought new optimism to the field. This study employed envelope-based immunogens delivered as a priming vaccination with a recombinant poxvirus vector and boosting with recombinant proteins. This regimen provided modest protection to HIV-1 infection in a low-risk population. Although the correlates of protection are currently unknown, extensive studies are underway to try to determine these. Neutralizing antibodies were not induced in the RV144 study; however, considerable titres of binding antibodies to HIV-1 viral envelope (Env) were. It is speculated that these antibodies may have provided a means of protection by a mechanism such as antibody-dependent cell-mediated cytotoxicity. In addition, no CD8+ T-cell responses were induced, but robust CD4+ T-cell responses were, and correlates of protection are being sought by analysing the quality of this aspect of the vaccine-induced immune response. The current paradigm for an optimal HIV-1 vaccine is to design immunogens and vaccination protocols that allow the induction of both broadly neutralizing humoral and broadly reactive and effective cell-mediated immunity, to act at sites of possible infection and post-infection, respectively. However, this is challenged by the results of the RV144 trial as neither of these responses were induced but modest protection was observed. Understanding the biology and immunopathology of HIV-1 early following infection, its modes of transmission and the human immune system's response to the virus should aid in the rational design of vaccines of increased efficacy.

A randomized phase II study of flutamide with or without PSA-TRICOM in nonmetastatic castration-resistant prostate cancer (CRPC).

163 Background: PSA-TRICOM is a vector-based, therapeutic cancer vaccine regimen consisting of recombinant poxviruses containing the transgenes for prostate-specific antigen (PSA) and 3 T-cell co-stimulatory molecules (TRICOM). A previous randomized, placebo-controlled phase II study demonstrated an 8.5-month improvement in median survival (25.1 months for PSA-TRICOM group vs. 16.6 months for control group) in men with metastatic CRPC. Methods: This study is currently enrolling patients with non-metastatic CRPC on testosterone suppression therapy who have a rising PSA. Patients are stratified by PSA doubling time and randomized to androgen receptor antagonist alone (flutamide) or flutamide plus PSA-TRICOM. Flutamide is given at the standard dose of 400 mg TID while PSA-TRICOM is given by monthly subcutaneous injections. The primary endpoint of the study is time to treatment failure (TTF) which is defined as biochemical recurrence (PSA rise) or development of metastatic lesions on scans. Results: The first 26 patients enrolled are evaluated in this analysis. For flutamide alone (n = 13), the median age at enrollment was 64.7 years and median Gleason Score was 8. For flutamide + PSA-TRICOM (n = 13), the median age at enrollment was 67.1 years and median Gleason score was 8. Median time to progression is 223 days for Fluatmide + PSA- TRICOM (range 70-638) vs. 85 days for Flutamide alone (56-372). Progression for 11/12 flutamide alone patients and 9/10 fluatmide + PSA-TRICOM patients has been by PSA rise only. Conclusions: Preliminary evidence suggests improvement in time to treatment failure using combination of hormonal therapy with flutamide + PSA-TRICOM vaccine compared to fluatmide alone in patients with non-metastatic CRPC. This trial will continue to accrue a total of 62 patients and also will evaluate immunological responses. No significant financial relationships to disclose.

Intraprostatic vaccine administration in patients with locally recurrent prostate cancer.

141 Background: PSA-TRICOM is a cancer vaccine consisting of recombinant poxviruses with transgenes for PSA and 3 T cell costimulatory molecules (TRICOM). A multicenter randomized vector controlled phase II study showed an 8.5 month improvement in median survival in men with metastatic CRPC. Preclinical studies showed intratumoral administration of TRICOM vaccines may improve anti-tumor outcomes. This study was intended to show safety of intraprostatic (i.p.) vaccine administration. 2° goals included immunologic and tumor response. Methods: 21 pts were enrolled with locally recurrent prostate cancer after radiation into 5 cohorts and received initial vaccination with s.c. recombinant vaccinia (rV)-PSA-TRICOM and booster i.p. recombinant fowlpox (rF)-PSA-TRICOM. Cohorts 3-5 also received i.p. rF-GM-CSF. Cohort 5 received concurrent s.c. and i.p. boosters. Priming was given on day 1, and boosters were given on days 29, 57, and 85. Pts had pre- and post-treatment prostate biopsies. Tumor infiltrate was evaluated when adequate tissue was available. PBMCs were analyzed by ELISPOT for response to PSA and 3-color flow cytometry to characterize regulatory T cells (Treg) or effector T cells. Treg suppression function was assayed by suppression of the CD4+ effector T cell proliferation. Results: Only 1 grade 3 toxicity occurred, a transient fever. Most common grade 2 adverse events for all doses were fever (14.5%) and injection-site reactions (21%). 15 of 21 pts had stable or improved PSA on study, and 16 of 21 pts had stable or improved PSA doubling time. Post-vaccination, 4 of 8 evaluable pts had immunological response by ELISPOT assay and 11 of 15 pts had decreased (n=8) or stable (n=3) Treg function. A paired t-test of 13 biopsies pre- and post- vaccination showed significant increases in immune infiltrates within tumors after vaccination. CD4+ cells increased from 1.3 → 13.1/high power field (hpf). CD8+ cells rose from 6.4 → 14/hpf. Conclusions: I.p. administration of PSA-TRICOM is safe, feasible and can generate a substantial immunologic response. Improved PSA kinetics and intense post-vaccination immune infiltrates were seen in the majority of pts. Trials of i.p. PSA-TRICOM examining clinical endpoints are warranted. No significant financial relationships to disclose.

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 dGag 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.

Engineering recombinant poxviruses using a compact GFP–blasticidin resistance fusion gene for selection

Recombinant poxviruses are important tools for research and some are candidate vaccines. To make these viruses a simple, small vector that can be used to engineer multiple strains of vaccinia virus and other model poxviruses, including ectromelia virus is of value. Here a set of plasmids and methods for making these viruses that uses an enhanced green fluorescent protein–blasticidin resistance (GFP–bsd) fusion gene as a transient selectable marker are described. This gene is smaller than any of the bi-functional selection markers used previously. The versatility of the method across different poxviruses is demonstrated by engineering changes into multiple loci of the WR and Modified Vaccinia Ankara (MVA) strains of vaccinia virus and also ectromelia virus. Finally, a set of vaccinia virus sequences for directing homologous recombination that are very highly conserved was designed and tested. These sequences allow a single plasmid to be used to insert a transgene into multiple strains of the virus.

Evaluation of poxviruses targeting the tumor microenvironment for cancer therapy.

2600 Background: The tumor microenvironment plays a critical role in mediating anti-tumor immunity following immunotherapy. To overcome the suppressive influence of the microenvironment, recombinant vaccinia and fowlpox viruses were designed to express B7.1 alone or a triad of costimulatory molecules, including B7.1, ICAM-1 and LFA-3 (TRICOM) for direct intra-lesional injection into tumors. The safety profile, immune response and therapeutic efficacy of the four poxviruses are reported herein. Methods: A series of phase I clinical trials were conducted in metastatic cancer patients who have measurable, superficial metastatic lesions (2 colorectal, 35 melanoma). Index tumors were injected with vaccinia virus expressing B7.1 (rV-B7.1, n = 12) or TRICOM (rV-TRICOM, n = 13) or fowlpox virus expressing B7.1 (rF-B7.1, n = 6) or TRICOM (rF-TRICOM, n = 6). Patients were evaluated for toxicity using NCI CTC and clinical response by standard RECIST criteria. Blood and tumor biopsies were collected for analysis of systemic and local immune response of vaccines. Results: There were no serious adverse events in any patient. The three most common adverse events were grade I fever (n = 13), grade I fatigue (n = 13) and grade I injection site pain (n = 16). All viruses induced significant anti-viral antibody titers following intra-tumoral injection, although rV- B7.1 induced the strongest response (2.9 fold increase) and rF-TRICOM the weakest (1.8 fold increase). Although all four viruses induced viral-specific T cell responses, both vaccinia vectors showed the strongest tumor-antigen specific T-cell responses. Objective clinical responses were observed in 3 of 12 (25%) patients treated with rV-B7.1, 5 of 13 (38%) with rV-TRICOM, 0 of 6 with rF-B7.1 and 0 of 6 with rF-TRICOM. Conclusions: Intratumoral injection of recombinant poxviruses is safe and capable of generating tumor-specific immunity in metastatic cancer patients. Vaccinia virus demonstrated better antigen-specific T-cell responses and therapeutic effectiveness in Phase I trials. Further studies using vaccinia vectors in combination with other agents or in prime-boost strategies with fowlpox virus might be considered for targeting the tumor microenvironment. No significant financial relationships to disclose.

Safety and Immunogenicity of Novel Recombinant BCG and Modified Vaccinia Virus Ankara Vaccines in Neonate Rhesus Macaques

Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA(401) or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA(401) induced high frequencies of BCG-specific IFN-gamma-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-gamma responses. MVA.HIVA elicited HIV-1-specific IFN-gamma responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.

Development and Evaluation of an Avian Influenza, Neuraminidase Subtype 1, Indirect Enzyme-Linked Immunosorbent Assay for Poultry Using the Differentiation of Infected from Vaccinated Animals Control Strategy

An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus, purified, recombinant N1 protein from A/chicken/Indonesia/PA7/2003 (H5N1) virus. The N1-ELISA showed high selectivity for detection of N1 antibodies, with no cross-reactivity with other neuraminidase subtypes, and broad reactivity with sera to N1 subtype isolates from North American and Eurasian lineages. Sensitivity of the N1-ELISA to detect N1 antibodies in turkey sera, collected 3 wk after H1N1 vaccination, was comparable to detection of avian influenza antibodies by the commercial, indirect ELISAs ProFLOK AIV Plus ELISA Kit (Synbiotics, Kansas City, MO) and Avian Influenza Virus Antibody Test Kit (IDEXX, Westbrook, ME). However, 6 wk after vaccination, the Synbiotics ELISA kit performed better than the N1-ELISA and the IDEXX ELISA kit. An evaluation was made of the ability of the N1-ELISA to discriminate vaccinated chickens from subsequently challenged chickens. Two experiments were conducted, chickens were vaccinated with inactivated H5N2 and H5N9 viruses and challenged with highly pathogenic H5N1 virus, and chickens were vaccinated with recombinant poxvirus vaccine encoding H7 and challenged with highly pathogenic H7N1 virus. Serum samples were collected at 14 days postchallenge and tested by hemagglutination inhibition (HI), quantitative neuraminidase inhibition (NI), and N1-ELISA. At 2 days postchallenge, oropharyngeal swabs were collected for virus isolation (VI) to confirm infection. The N1-ELISA was in fair agreement with VI and HI results. Although the N1-ELISA showed a lower sensitivity than the NI assay, it was demonstrated that detection of N1 antibodies by ELISA was an effective and rapid assay to identify exposure to the challenge virus in vaccinated chickens. Therefore, N1-ELISA can facilitate a vaccination strategy with differentiation of infected from vaccinated animals using a neuraminidase heterologous approach.

Consumption of Baits Containing Raccoon Pox-Based Plague Vaccines Protects Black-Tailed Prairie Dogs (Cynomys ludovicianus)

Baits containing recombinant raccoon poxvirus (RCN) expressing plague antigens (fraction 1 [F1] and a truncated form of the V protein-V307) were offered for voluntary consumption several times over the course of several months to a group of 16 black-tailed prairie dogs (Cynomys ludovicianus). For comparison, another group of prairie dogs (n = 12) was injected subcutaneously (SC) (prime and boost) with 40 microg of F1-V fusion protein absorbed to alum, a vaccine-adjuvant combination demonstrated to elicit immunity to plague in mice and other mammals. Control animals received baits containing RCN without the inserted antigen (n = 8) or injected diluent (n = 7), and as there was no difference in their survival rates by Kaplan-Meier analysis, all of them were combined into one group in the final analysis. Mean antibody titers to Yersinia pestis F1 and V antigen increased (p < 0.05) in the vaccinated groups compared to controls, but titers were significantly higher (p < 0.0001) in those receiving injections of F1-V fusion protein than in those orally vaccinated with RCN-based vaccine. Interestingly, upon challenge with approximately 70,000 cfu of virulent Y. pestis, oral vaccination resulted in survival rates that were significantly higher (p = 0.025) than the group vaccinated by injection with F1-V fusion protein and substantially higher (p < 0.0001) than the control group. These results demonstrate that oral vaccination of prairie dogs using RCN-based plague vaccines provides significant protection against challenge at dosages that simulate simultaneous delivery of the plague bacterium by numerous flea bites.

Immunogenicity of viral vector, prime-boost SIV vaccine regimens in infant rhesus macaques: Attenuated vesicular stomatitis virus (VSV) and modified vaccinia Ankara (MVA) recombinant SIV vaccines compared to live-attenuated SIV

In a previously developed infant macaque model mimicking HIV infection by breast-feeding, we demonstrated that intramuscular immunization with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins provided partial protection against infection following oral inoculation with virulent SIV. In an attempt to further increase systemic but also local antiviral immune responses at the site of viral entry, we tested the immunogenicity of different orally administered, replicating vaccines. One group of newborn macaques received an oral prime immunization with a recombinant vesicular stomatitis virus expressing SIVmac239 gag, pol and env (VSV-SIVgpe), followed 2 weeks later by an intramuscular boost immunization with MVA-SIV. Another group received two immunizations with live-attenuated SIVmac1A11, administered each time both orally and intravenously. Control animals received mock immunizations or non-SIV VSV and MVA control vectors. Analysis of SIV-specific immune responses in blood and lymphoid tissues at 4 weeks of age demonstrated that both vaccine regimens induced systemic antibody responses and both systemic and local cell-mediated immune responses. The safety and immunogenicity of the VSV-SIVgpe+MVA-SIV immunization regimen described in this report provide the scientific incentive to explore the efficacy of this vaccine regimen against virulent SIV exposure in the infant macaque model.

Canine Distemper Vaccination is a Safe and Useful Preventive Procedure for Southern Sea Otters (Enhydra lutra nereis)

From 2002 to 2006, eight captive southern sea otters (Enhydra lutris nereis) at research and display institutions in California at risk of exposure to potentially lethal morbiliviruses were vaccinated with a commercial recombinant poxvirus vectored canine distemper (CD) vaccine. Serum-neutralizing (SN) antibody responses were followed for several years. The goal of this study was to determine whether 1) CD vaccination was a safe preventive medicine procedure for this species; 2) sea otters produce detectable SN antibodies in response to vaccination with this product; and 3) if this type of vaccination might be useful in response to a morbilivirus disease outbreak in free-ranging sea otters. Results indicate that a commercial recombinant vaccine is safe, provokes a measurable SN antibody response, and that vaccination may provide some protection from infection for free-ranging sea otters. It also resulted in the reevaluation of CD serology data that were previously published for free-ranging sea otters. Canine distemper, Enhydra lutris nereis, morbilivirus, sea otter, serology, vaccination.

Concurrent vaccination with two distinct vaccine platforms targeting the same antigen generates phenotypically and functionally distinct T-cell populations

Studies comparing two or more vaccine platforms have historically evaluated each platform based on its ability to induce an immune response and may conclude that one vaccine is more efficacious than the other(s), leading to a recommendation for development of the more effective vaccine for clinical studies. Alternatively, these studies have documented the advantages of a diversified prime and boost regimen due to amplification of the antigen-specific T-cell population. We hypothesize here that two vaccine platforms targeting the same antigen might induce shared and distinct antigen-specific T-cell populations, and examined the possibility that two distinct vaccines could be used concomitantly. Using recombinant poxvirus and yeast vaccines, we compared the T-cell populations induced by these two platforms in terms of serum cytokine response, T-cell gene expression, T-cell receptor phenotype, antigen-specific cytokine expression, T-cell avidity, and T-cell antigen-specific tumor cell lysis. These studies demonstrate for the first time that vaccination with a recombinant poxvirus platform (rV/F-CEA/TRICOM) or a heat-killed yeast vaccine platform (yeast-CEA) elicits T-cell populations with both shared and unique phenotypic and functional characteristics. Furthermore, both the antigen and the vector play a role in the induction of distinct T-cell populations. In this study, we demonstrate that concurrent administration of two vaccines targeting the same antigen induces a more diverse T-cell population that leads to enhanced antitumor efficacy. These studies provide the rationale for future clinical studies investigating concurrent administration of vaccine platforms targeting a single antigen to enhance the antigen-specific immune response.

Identification of Ongoing Human Immunodeficiency Virus Type 1 (HIV-1) Replication in Residual Viremia during Recombinant HIV-1 Poxvirus Immunizations in Patients with Clinically Undetectable Viral Loads on Durable Suppressive Highly Active Antiretroviral Therapy

In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of <50 copies/ml during highly active antiretroviral therapy (HAART), low levels of plasma virus remain detectable for years by ultrasensitive methods. The relative contributions of ongoing virus replication and virus production from HIV-1 reservoirs to persistent low-level viremia during HAART remain controversial. HIV-1 vaccination of HAART-treated individuals provides a model for examining low-level viremia, as immunizations may facilitate virus replication and sequence evolution. In a phase 1 trial of modified vaccinia virus Ankara/fowlpox virus-based HIV-1 vaccines in 20 HIV-infected young adults receiving HAART, we assessed the prevalence of low-level viremia and sequence evolution, using ultrasensitive viral load (<6.5 copies/ml) and genotyping (five-copy sensitivity) assays. Viral evolution, consisting of new drug resistance mutations and novel amino acid changes within a relevant HLA-restricted allele (e.g., methionine, isoleucine, glutamine, or arginine for leucine at position 205 of RT), was found in 1 and 3 of 20 subjects, respectively. Sequence evolution was significantly correlated with levels of viremia of between 6.5 and <50 copies/ml (P = 0.03) and was more likely to occur within epitopes presented by relevant HLA alleles (P < 0.001). These findings suggest that ongoing virus replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject to CD8(+) T-cell selective pressures.

Harnessing the unique local immunostimulatory properties of modified vaccinia Ankara (MVA) virus to generate superior tumor-specific immune responses and antitumor activity in a diversified prime and boost vaccine regimen

Recombinant poxviruses expressing tumor-associated antigens (TAAs) are currently being evaluated in clinical trials as an approach to treat various cancers. We have previously generated poxviral vectors expressing a TAA and a TRIad of COstimulatory Molecules (B7-1, ICAM-1, and LFA-3; TRICOM) as transgenes, including replication competent recombinant vaccinia (rV) or replication-defective modified vaccinia Ankara (MVA), to prime tumor-specific immune responses, and a replication-defective recombinant fowlpox (rF) to boost these responses. MVA is a potentially safer, replication-defective form of vaccinia virus with unique immunostimulatory properties that could make it a superior priming vaccine. Here, an MVA vector encoding a tumor antigen (CEA) and TRICOM was utilized (rMVA). A single rMVA-CEA/TRICOM vaccination induced greater expression of several serum cytokines associated with enhanced T-cell immunity than that seen with vaccinia. We hypothesized that this effect might “precondition” the vaccination site for a more effective boost. An rMVA-CEA/TRICOM prime followed 7 days later (but not 30 days later) by an rF-CEA/TRICOM boost at the same injection site (but not at a distal site) induced more potent CEA-specific T-cell responses, and superior CEA-specific immunity and antitumor activity, than rV-CEA/TRICOM followed by rF-CEA/TRICOM. This preconditioning effect was also observed using a heterologous antigen model, where priming with rMVA-CEA/TRICOM followed 7 days later by rF-LacZ/TRICOM enhanced β-gal-specific immunity compared to rF-LacZ/TRICOM only. The studies reported here show for the first time that priming with rMVA followed 7 days later by an rF boost at the same injection site, versus a distal site, generates superior tumor-specific immunity and antitumor activity.