Robert L. Wilson

Genetic immunization in the lung induces potent local and systemic immune responses

Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.

Nasal DNA-MVA SIV vaccination provides more significant protection from progression to AIDS than a similar intramuscular vaccination.

Preventive human immunodeficiency virus (HIV) vaccination may require induction of virus-specific immune responses at mucosal sites to contain viral infection locally after exposure, as most HIV infections occur through mucosal surfaces. We compared the efficacy of an intranasal or intramuscular Simian immunodeficiency virus (SIV)+ interleukin (IL)-2+IL-15 DNA/SIV–MVA (modified vaccinia virus Ankara) vaccination in preventing disease progression in SIVmac251 intrarectally challenged rhesus macaques. SIV-specific rectal IgA responses were more significantly persistent in nasally vaccinated than in intramuscularly vaccinated animals. No significant differences were observed in the magnitude of systemic T-cell responses between the two groups, although the nasal immunization induced more significant anti-SIV T-cell responses in the colorectal mucosa. After challenge, CD4+ central memory (CM) T-cell preservation and significant disease-delay were observed in both vaccination groups. However, nasally vaccinated animals had more significant early preservation of circulating and colorectal CD4+ CM T cells, of circulating CD4+/α4β7+ effector memory (EM) T cells, and a longer disease-free interval when compared with the intramuscularly vaccinated or control groups. Regardless of vaccination status, long-term viremia control and preservation of CD4+ CM T cells was detected in animals with significantly higher systemic CD8+/tumor necrosis factor (TNF)-α+ and CD8+/interferon (IFN)-γ+ T-cell responses and higher SIV-specific CD4+/IL-2+ responses in colorectal T cells.

DNA-MVA vaccine protection after X4 SHIV challenge in macaques correlates with day-of-challenge antiviral CD4+ cell-mediated immunity levels and postchallenge preservation of CD4+ T cell memory

The ability of vaccines to induce immunity both in mucosal and systemic compartments may be required for prevention of HIV infection and AIDS. We compared DNA-MVA vaccination regimens adjuvanted by IL-12 DNA, administered intramuscularly and nasally or only nasally. Most of the vaccinated Rhesus macaques developed mucosal and systemic humoral and cell-mediated SHIV-specific immune responses. Stimulation of mucosal anti-Env IgA responses was limited. After rectal challenge with SHIV 89.6P, all vaccinated and naive animals became infected. However, most of the vaccinated animals showed significant control of viremia and protection from CD4(+) T cell loss and AIDS progression compared to the control animals. The levels of CD4(+) and CD8(+) T cell virus-specific responses measured on the day of challenge correlated with the level of viremia control observed later during the chronic infection. Postchallenge viremia levels inversely correlated with the preservation of SHIV-specific CD4(+)/IL-2(+) and CD8(+)/TNF-alpha(+) T cells but not with CD4(+)/IFN-gamma(+) T cells measured over time after challenge. We also found that during the early chronic infection SHIV vaccination permitted a more significant preservation of both naive and memory CD4(+) T cells compared to controls. In addition, we observed a more significant and prolonged preservation of memory CD4(+) T cells after SHIV vaccination and challenge than that observed after SIV vaccination and challenge. As the antiviral immunity stimulated by vaccination is present in the memory CD4(+) T cell subpopulations, its more limited targeting by SHIV compared to SIV may explain the better control of X4 tropic SHIV than R5 tropic SIVs by vaccination.

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.

Preexisting Vaccinia Virus Immunity Decreases SIV-Specific Cellular Immunity but does not diminish Humoral Immunity and Efficacy of a DNA/MVA Vaccine

The influence of preexisting immunity to viral vectors is a major issue for the development of viral-vectored vaccines. In this study, we investigate the effect of preexisting vaccinia virus immunity on the immunogenicity and efficacy of a DNA/modified vaccinia Ankara (MVA) SIV vaccine in rhesus macaques using a pathogenic intrarectal SIV251 challenge. Preexisting immunity decreased SIV-specific CD8 and CD4 T cell responses but preserved the SIV-specific humoral immunity. In addition, preexisting immunity did not diminish the control of an SIV challenge mediated by the DNA/MVA vaccine. The peak and set point viremia was 150- and 17-fold lower, respectively, in preimmune animals compared with those of control animals. The peak and set point viremia correlated directly with colorectal virus at 2 wk postchallenge suggesting that early control of virus replication at the site of viral challenge was critical for viral control. Factors that correlated with early colorectal viral control included 1) the presence of anti-SIV IgA in rectal secretions, 2) high-avidity binding Ab for the native form of Env, and 3) low magnitude of vaccine-elicited SIV-specific CD4 T cells displaying the CCR5 viral coreceptor. The frequency of SIV-specific CD8 T cells in blood and colorectal tissue at 2 wk postchallenge did not correlate with early colorectal viral control. These results suggest that preexisting vaccinia virus immunity may not limit the potential of recombinant MVA vaccines to elicit humoral immunity and highlight the importance of immunodeficiency virus vaccines achieving early control at the mucosal sites of challenge.

Long-Term Control of Simian Immunodeficiency Virusmac251 Viremia to Undetectable Levels in Half of Infected Female Rhesus Macaques Nasally Vaccinated with Simian Immunodeficiency Virus DNA/Recombinant Modified Vaccinia Virus Ankara

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIVmac251 low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4β7high+/CD4+ T cells and of circulating and mucosal CD4+/CM T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4+/IFN-γ+ and CD8+/IFN-γ+ T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4+/IL-2+, CD4+/IFN-γ+, and CD8+/TNF-α+ T cells and vaginal anti-SIV Gag + Env CD8+ T cell total monofunctional responses.

Resistance to Infection, Early and Persistent Suppression of Simian Immunodeficiency Virus SIVmac251 Viremia, and Significant Reduction of Tissue Viral Burden after Mucosal Vaccination in Female Rhesus Macaques

ABSTRACT The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIVmac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIVmac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8+ T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4+ central memory and CD4+/α4β7+ T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4+, and CD8+ T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.

Envelope-Modified Single-Cycle Simian Immunodeficiency Virus Selectively Enhances Antibody Responses and Partially Protects against Repeated, Low-Dose Vaginal Challenge

ABSTRACT Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIVmac239 or SIVmac251UCD, neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIVmac251UCD, six of eight immunized animals versus six of six naïve controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naïve control animals.

OA07-03. Influence of preexisting vaccinia immunity on a DNA/MVA SIV vaccine, decreased cellular immunity but enhanced control of a pathogenic SIV challenge

Methods Three groups of rhesus macaques, eight per group, were studied. The Dryvax-naive and Dryvax-immune groups received the DNA/MVA SIV vaccine (DNA at weeks 0 and 8, and rMVA at weeks 16 and 24). In addition, the Dryvaximmune group received the Dryvax vaccine 1.5 years prior to the DNA prime. The control group did not receive any vaccine. All macaques were challenged intrarectally with SIV251 at 9 months after the final MVA.