Multigenic DNA Vaccine Induces Protective Cross-Reactive T Cell Responses Against Heterologous Influenza Virus in Nonhuman Primates

Recent avian and swine-origin influenza virus outbreaks illustrate the ongoing threat of influenza pandemics. We investigated immunogenicity and protective efficacy of a multi-antigen (MA) universal influenza DNA vaccine consisting of HA, M2, and NP antigens in cynomolgus macaques. Following challenge with a heterologous pandemic H1N1 strain, vaccinated animals exhibited significantly lower viral loads and more rapid viral clearance when compared to unvaccinated controls. The MA DNA vaccine induced robust serum and mucosal antibody responses but these high antibody titers were not broadly neutralizing. In contrast, the vaccine induced broadly-reactive NP specific T cell responses that cross-reacted with the challenge virus and inversely correlated with lower viral loads and inflammation. These results demonstrate that a MA DNA vaccine that induces strong cross-reactive T cell responses can, independent of neutralizing antibody, mediate significant cross-protection in a nonhuman primate model and further supports development as an effective approach to induce broad protection against circulating and emerging influenza strains

Correction: Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

[This corrects the article DOI: 10.1371/journal.pone.0198154.]

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation

<div><p>A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.</p></div

A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response.

Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza

Multigenic DNA vaccine induces protective cross-reactive T cell responses against heterologous influenza virus in nonhuman primates.

Recent avian and swine-origin influenza virus outbreaks illustrate the ongoing threat of influenza pandemics. We investigated immunogenicity and protective efficacy of a multi-antigen (MA) universal influenza DNA vaccine consisting of HA, M2, and NP antigens in cynomolgus macaques. Following challenge with a heterologous pandemic H1N1 strain, vaccinated animals exhibited significantly lower viral loads and more rapid viral clearance when compared to unvaccinated controls. The MA DNA vaccine induced robust serum and mucosal antibody responses but these high antibody titers were not broadly neutralizing. In contrast, the vaccine induced broadly-reactive NP specific T cell responses that cross-reacted with the challenge virus and inversely correlated with lower viral loads and inflammation. These results demonstrate that a MA DNA vaccine that induces strong cross-reactive T cell responses can, independent of neutralizing antibody, mediate significant cross-protection in a nonhuman primate model and further supports development as an effective approach to induce broad protection against circulating and emerging influenza strains

Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.

<p>Rapamycin alters the cytokine profile in the cerebrospinal fluid after gene delivery.</p

GFP-specific T cell responses as spot forming cells (SFC)/10⁶ PBMC after rapamycin treatment.

<p>T cell responses in PBMC were measured by ELISPOT at 14, 28, and 84 days after AAV9/GFP delivery in (A) AAV9/GFP only (controls); (B) AAV9/GFP + rapamycin. Arrow indicates undetectable (< 10 SFC/10⁶ PBMC) GFP-specific responses at necropsy (84 days).</p

IHC was carried out to visualize GFP expression in the lumbar spinal cord.

<p>Shown are representative 40 micron lumbar spinal cord sections of all study macaques, stained for GFP. Magnified insets show examples of GFP-positive motor neurons. Macaque ID numbers are provided in each panel, along with a qualitative scoring of ventral horn expression. The (-) indicates no GFP-positive staining above that seen in uninjected NHPs; (+) indicates some positive cells observed, above that seen in uninjected macaques; (++) indicates relatively strong expression in a large number of neurons.</p

GFP expression in lumbar spinal cords of NHPs monitored by IHC.

<p>GFP expression in lumbar spinal cords of NHPs monitored by IHC.</p

Cytotoxic GFP-specific T cells decreased after rapamycin treatment.

<p>Multiparameter flow cytometry was used to identify CD8+ CD107+ T cells in PBMC after stimulation with GFP peptide pools. CD8+CD107+ expression in PBMC was monitored 28 and 84 days after transgene delivery in (A) AAV9/GFP (controls); (B) AAV9/GFP and rapamycin.</p