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Studies focused on the understanding of the molecular mechanisms involved in recovery or progression to chronicity of HBV may take advantage of natural and experimental models that mimic its properties. This is also of relevance for associated diseases such as cirrhosis and hepatocellulocarcinoma. The eastern woodchuck (Marmota monax) infected by the hepadnavirus woodchuck Hepatitis B virus (WHV) has been applied as a predictive model to support development of new HBV vaccines, antivirals, immunotherapies and combination therapies. This report summarizes studies carried out by our and other groups, with the application of this model in natural and experimental infections. Using standardized viral inocula in neonate and adult animals and newly established assays, the presence of the specific patterns of markers of acute, chronic and resolved infections and their relationships in the different virus-host interactions have been shown. B and T cell responses and T(H)1 cytokine expression have been shown to play a crucial role in the outcome of infection. The availability of the WHV/Marmota monax model and specific standardized assays may allow evaluation of new formulations of multimodal therapeutic strategies based on antiviral chemotherapy and immunomodulation. These may also include specifically targeted immunocomplexes. Such therapies could constitute new frontiers for the treatment of HBV chronic disease.

The woodchuck hepatitis B virus (WHV), the closest genetically related virus of HBV, and its natural host Marmota monax constitute a well recognized animal model. The application of this model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use, for lamivudine-CHO-PreS/S vaccine therapy and WHV particles coupled with HBV derived woodchuck PreS/S antibodies (IC complex) studies, as shown that the PreS/S-CHO vaccine is the first human vaccine able to elicit non sterilizing protection in the woodchuck model. The very early appearance and selection of the domain B FLLA motif resistant mutant not neutralized by the antibodies produced following vaccination, has confirmed that more potent antivirals and/or multiple targeted options with possible inclusion of immune complexes should be considered.

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.

Several studies have shown the importance of evaluating Recent Thymic Emigrants (RTEs) by quantification of T cell receptor-rearrangement excision circles (TRECs), as a measure of de novo T cell generation during human immunodeficiency virus-1 (HIV-1) infection. To determine whether acute viral infection may have an impact on TRECs, cynomolgus monkeys (Macaca fascicularis) were infected intrarectally with simian human immunodeficiency virus (SHIV) 89.6P(cy11) and the number of signal-joint (sj) TRECs was determined in purified CD4+ and CD8+ populations for up to 28 weeks post-infection. Four weeks after infection, TRECs levels significantly decreased in both CD3+ CD4+ and in CD3+ CD8+ T lymphocytes of infected monkeys, whereas they remained unchanged in uninfected animals. This reduction was followed by a progressive TRECs number recovery in CD3+ CD4+ T lymphocytes that positively correlated with changes in the levels of circulating CD3+ CD4+ T cells. In the CD3+ CD8+ T cell subset, TRECs number remained significantly low and inversely correlated with the increase in the percentages of CD3+ CD8+ T cells. These data suggest that SHIV89.6P(cy11) intrarectal infection of cynomolgus monkeys differently affects TRECs content in CD3+ CD4+ and CD3+ CD8+ T cell subsets.

Alpha-defensins have been shown to inhibit HIV-1 replication in vitro and may contribute to the overall control of viral replication in vivo. In the present work, we quantitatively measured the levels of alpha-defensins in the plasma of healthy and experimentally SHIV-infected Macaca fascicularis (cynomolgus monkeys), an animal model of AIDS pathogenesis and vaccine development. Characterization of physiological plasma alpha-defensins levels was performed in 12 healthy monkeys following longitudinal analysis using an alpha-defensins ELISA kit currently validated for macaque use. Subsequently, alpha-defensins levels were quantitatively measured in 23 cynomolgus monkeys during titration protocols following both the mucosal and systemic routes of infection with the pathogenic SHIV89.6P(cy11). A significant increase in plasma alpha-defensins levels was consistently observed at early time points in all infected animals, regardless of the infection route. Moreover, a positive correlation was observed between viral replication and levels of alpha-defensins during the acute phase of infection. Interestingly, in the animals infected through the mucosal route, alpha-defensins levels remained significantly higher at later time points, up to 19 weeks from the infection, while in cynomolgus infected intravenously, alpha-defensins levels returned to baseline levels by 4 weeks from infection, suggesting that the different route of infection may differently activate the innate immune response.

We investigated the protective efficacy of a systemic triple vector (DNA/rSFV/rMVA)-based vaccine against mucosal challenge with pathogenic simian immunodeficiency virus (SIV) in cynomolgus monkeys. Animals were immunized at week 0 with DNA (intradermally), at weeks 8 and 16 with recombinant Semliki Forest virus (rSFV, subcutaneously) and finally, at week 24, with recombinant modified vaccinia virus Ankara strain (rMVA, intramuscularly). Both DNA and recombinant viral vectors expressed a wide range of SIV proteins (Gag, Pol, Tat, Rev, Env and Nef). This immunization strategy elicited cell-mediated rather than humoral responses that were especially increased following the last boost. Upon intrarectal challenge with pathogenic SIVmac251, three of the four vaccinated monkeys dramatically abrogated virus load to undetectable levels up to 41 weeks after challenge. A major contribution to this vaccine effect appeared to be the T-cell-mediated immune response to vaccine antigens (Gag, Rev, Tat, Nef) seen in the early phase of infection in three of the four vaccinated monkeys. Indeed, the frequency of T-cells producing antigen-induced IFN-γmirrored virus clearance in the vaccinated and protected monkeys. These results, reminiscent of the efficacy of live attenuated virus vaccines, suggest that vaccination with a combination of many viral antigens can induce a robust and stable vaccine-induced immunity able to abrogate virus replication.

The Eastern woodchuck (Marmota monax) is a useful experimental model for evaluating antiviral therapy against chronic HBV infection. In the present study, an immunogenic complex (IGC) composed of immune sera containing PreS/S heterologous antibodies (anti-HBs) and serum-derived WHV particles containing 10(7) WHV-DNA copies/50 µl was developed. The IGC was administered to WHV-negative woodchucks and natural chronic WHV carriers, with the final aim of evaluating the outcome of WHV infection in both groups. A control group of three animals, infected experimentally with viral particles only, was also evaluated. Following IGC administration, two WHV-negative woodchucks exhibited persistent infection, with WHV-DNA levels 3-6 logs lower than the WHV-DNA levels of the controls that developed persistent infection. WHeAg seroconversion to anti-WHe was observed in these two woodchucks and in two control woodchucks which developed self-limited infection. In two of the four chronic carriers, the WHV-DNA level decreased significantly (by 4-6 logs) following IGC administration, with no rebound in viral load during follow-up. WHeAg seroconversion to anti-WHe was observed also in these animals. Analyses of the sequences derived from envelope proteins confirmed that IGC did not induce the emergence of resistant viral variants. The results of this study indicate that the IGC could be useful for breaking the tolerance in hepadnaviral infection and for boosting the host's innate and adoptive immune response.

Present efforts of HBV vaccine research are aimed at defining targeted antigen compositions and adjuvancy systems for earlier and broader immune responses and optimization of immunotherapeutic approaches. We have demonstrated the applicability of the WHV/Marmota monax model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use. Protective activity was evaluated following the administrations of HBV CHO-PreS/S and adjuvanted S/Core vaccines. The administration of a complex constituted by HBV derived woodchuck PreS/S antibodies coupled with WHV particles was able to induce inhibition of viral replication. Future studies on treatment of HBV chronic infection should be addressed to the evaluation of therapies combined with antivirals, vaccines and immunomodulatory compounds.

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIV(mac251/32H/J5)) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA-J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIV(mac251). All control monkeys, previously inoculated with non-recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post-challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA-J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell- and plasma-associated viral load.

The immunogenicity and the protective efficacy of a new polyvalent triple vector (DNA/SFV/MVA) based vaccine against mucosal challenge with pathogenic SIV m a c 2 5 1 were investigated. Cynomolgus monkeys (Macaca fascicularis) were primed intradermally with DNA, boosted twice subcutaneously with recombinant Semliki Forest virus (rSFV) and finally intramuscularly with recombinant Modified Vaccinia Virus Ankara strain (rMVA). Both DNA and recombinant viral vectors expressed SIV proteins (Gag, Pol, Tat, Rev, Nef and Env). The vaccinated monkeys developed T helper proliferative responses to viral antigens after the second immunization while interferon (IFN)-γ enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) specific responses appeared only after the last boost with rMVA. Upon intrarectal challenge with pathogenic SIV m a c 2 5 1 , three of four vaccinated monkeys were either fully protected or exhibited a dramatic reduction of virus replication up to undetectable level. A major contribution to this protective effect appeared to be the anamnestic T-cell IFN-γ ELISPOT responses to vaccine antigens (Gag, Rev, Tat, Nef) that mirrored the viral clearance. These results underline the efficacy of a multiprotein approach in combination with a triple vector system of antigen delivery.