Intranasal seasonal influenza vaccine and a TLR-3 agonist, rintatolimod, induced cross-reactive IgA antibody formation against avian H5N1 and H7N9 influenza HA in humans.

The intranasal use of rintatolimod, a specific TLR-3 agonist, combined with trivalent seasonal influenza vaccine generated cross-protection against highly pathogenic H5N1 avian influenza in mice. The purpose of this clinical trial is to assess the safety and impact of rintatolimod on intranasal influenza vaccine in healthy adults. During Stage I of this Phase I/II clinical trial, 12 volunteers were immunized intranasally with 3 doses of FluMist seasonal influenza vaccine on Days 0, 28, and 56 followed by intranasal rintatolimod (50 µg, 200 µg, or 500 µg) 3 days later. Parotid saliva and nasal wash samples were collected at baseline and on Days 25, 53, 84, and 417. The samples were tested for IgA and IgG specific antibodies (Ab) directed against the homologous FluMist viral hemagglutinins (HAs). In addition, viral specific responses against influenza A HAs were tested for IgA Ab cross-reactivity against 3 H5 clades: HA (H5N1) A/Indonesia/5/2005, HA (H5N1) A/Hong Kong/483/97 and HA (H5N1) A/Vietnam/1194/2004, as well as, two H7 strains, HA (H7N9) A/Shanghai/2/2013 and HA (H7N3) A/chicken/Jalisco/CPA1. The combination of the intranasal FluMist along with the rintatolimod generated specific secretory IgA responses of at least 4-fold over baseline against at least one of the homologous vaccine strains included in the vaccine in 92% of the vaccinees. Additionally, this vaccination strategy induced cross-reactive secretory IgA against highly pathogenic avian influenza virus strains H5N1, H7N9, and H7N3 with pandemic potential for humans. The combination of rintatolimod and FluMist was well-tolerated.

Protection from pulmonary tissue damage associated with infection of cynomolgus macaques by highly pathogenic avian influenza virus (H5N1) by low dose natural human IFN-α administered to the buccal mucosa.

Using an established nonhuman primate model for H5N1 highly pathogenic influenza virus infection in humans, we have been able to demonstrate the prophylactic mitigation of the pulmonary damage characteristic of human fatal cases from primary influenza virus pneumonia with a low dose oral formulation of a commercially available parenteral natural human interferon alpha (Alferon N Injection®). At the highest oral dose (62.5IU/kg body weight) used there was a marked reduction in the alveolar inflammatory response with minor evidence of alveolar and interstitial edema in contrast to the hemorrhage and inflammatory response observed in the alveoli of control animals. The mitigation of severe damage to the lower pulmonary airway was observed without a parallel reduction in viral titers. Clinical trial data will be necessary to establish its prophylactic human efficacy for highly pathogenic influenza viruses.

Sensitivity of SARS/MERS CoV to interferons and other drugs based on achievable serum concentrations in humans.

A novel coronavirus (MERS-CoV) related to SARS-CoV recently emerged in the Middle East causing more than 400 deaths with a mortality rate of about 30%, much higher than SARS-CoV. Both viruses target epithelial cells in the respiratory tract, although utilizing different cellular receptors. Because of the sporadic nature of the MERS outbreak and difficulty in collecting randomized, controlled clinical data, the objective of this review was to focus on published in vitro and in vivo drug sensitivity data using both cell lines and available animal models of SARS/MERS CoV infection. Determination of drug activity was based on achievable serum levels in humans relative to in vitro IC50 (50% inhibitory concentration) or EC50 (50% effective concentration) drug concentrations. The most active drugs against SARS/MERS CoV at clinically achievable serum levels were type I interferons and a TLR3 agonist, interferon inducer/activator.

Discordant biological and toxicological species responses to TLR3 activation.

Toll-like receptors (TLRs) are highly conserved type 1 membrane proteins that initiate a multiplicity of transient gene transcriptions, resulting in innate and adaptive immune responses. These essential immune responses are triggered by common TLR pattern recognition receptors of microbial products expressed through the cytoplasmic carboxy-terminal Toll/IL-1 domain. Toll/IL-1 adapter protein cascades are induced by an activated Toll/IL-1 to induce transient transcription responses. All TLRs, with the exception of TLR3, use an MyD88 adapter to Toll/IL-1 to initiate a proinflammatory cascade. TLR3 uses the toll receptor 3/4 induction factor adapter to initiate a different cytosolic adapter cascade with double-stranded RNA agonists. This non-MyD88 pathway induces both NF-κB and type 1 interferon responses. By using a TLR3-restricted double-stranded RNA agonist, rintatolimod, we demonstrate significant unexpected differences in toxic responses between rats and primates. The mechanism of this differential response is consistent with a relative down-regulation of the NF-κB inflammatory cytokine induction pathway in the cynomolgus monkey and humans, but not observed systemically in rat. Our findings suggest evaluation of TLR3 agonists in drug therapy.

Emergence of a novel drug resistant H7N9 influenza virus: evidence based clinical potential of a natural IFN-α for infection control and treatment.

The novel avian H7N9 influenza virus has caused more than 130 human infections with 43 deaths (as of September, 2013) in China. Because of the lack of existing immunity against H7 subtype influenza viruses in the human population and the absence of a licensed commercial vaccine, antiviral drugs are critical tools for the treatment of infection with this novel H7N9. Both M2-ion channel blockers and neuraminidase inhibitors are used as antiviral drugs for influenza infections of humans. The emerging H7N9 viruses are resistant to the M2-ion channel blockers because of a S31N mutation in the M2 protein; additionally, some H7N9 isolates have gained neuraminidase R292K substitution resulting in broad resistance to neuraminidase inhibitors. In this study we report that Alferon N can inhibit wild type and 292K H7N9 viruses replication in vitro. Since Alferon N is approved for clinical use, this would allow a rapid regulatory approval process for this drug under pandemic threat.

A double-blind, placebo-controlled, randomized, clinical trial of the TLR-3 agonist rintatolimod in severe cases of chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a severely debilitating disease of unknown pathogenesis consisting of a variety of symptoms including severe fatigue. The objective of the study was to examine the efficacy and safety of a TLR-3 agonist, rintatolimod (Poly I: C(12)U), in patients with debilitating CFS/ME. METHODS AND FINDINGS: A Phase III prospective, double-blind, randomized, placebo-controlled trial comparing twice weekly IV rintatolimod versus placebo was conducted in 234 subjects with long-standing, debilitating CFS/ME at 12 sites. The primary endpoint was the intra-patient change from baseline at Week 40 in exercise tolerance (ET). Secondary endpoints included concomitant drug usage, the Karnofsky Performance Score (KPS), Activities of Daily Living (ADL), and Vitality Score (SF 36). Subjects receiving rintatolimod for 40 weeks improved intra-patient placebo-adjusted ET 21.3% (p = 0.047) from baseline in an intention-to-treat analysis. Correction for subjects with reduced dosing compliance increased placebo-adjusted ET improvement to 28% (p = 0.022). The improvement observed represents approximately twice the minimum considered medically significant by regulatory agencies. The rintatolimod cohort vs. placebo also reduced dependence on drugs commonly used by patients in an attempt to alleviate the symptoms of CFS/ME (p = 0.048). Placebo subjects crossed-over to receive rintatolimod demonstrated an intra-patient improvement in ET performance at 24 weeks of 39% (p = 0.04). Rintatolimod at 400 mg twice weekly was generally well-tolerated. CONCLUSIONS/SIGNIFICANCE: Rintatolimod produced objective improvement in ET and a reduction in CFS/ME related concomitant medication usage as well as other secondary outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00215800.

Recombinant and natural human interferons: analysis of the incidence and clinical impact of neutralizing antibodies.

This review summarizes and analyzes the clinical outcomes following treatment of a wide range of diseases with recombinant interferons (r-IFNs) and/or natural interferons (n-IFNs). The investigation focuses on the frequency of neutralizing antibodies (NABs) directed against IFN, which are formed during treatment and their clinical impact. r-IFNs (α-2a, α-2b, ß-1a, and ß-1b) induced seroconversion with generation of NABs in 17.2% of patients studied. The highest incidence of NABs occurred in macular degeneration (61.4%) with the lowest in multiple sclerosis (14.7%). The incidence of antibodies induced against n-IFNs was very low (<0.2%) and was significantly less than that seen for r-IFNs (P<0.0001). Overall, the fraction of relapsed and refractory patients is statistically greater in NAB positive patients compared to NAB negative patients (<0.0001), whereas the percentage of responding patients is higher in the NAB negative cohort (P<0.001). Finally, we also analyzed relapsed and refractory NAB positive patients who switched treatment to n-IFN, such as leukocyte derived Alferon N Injection® (α-n3) or Wellferon® (α-n1). Overall, in 33/40 (82%) of these relapsed or refractory patients, switching to n-IFNs restored the clinical response. This result is consistent with serology studies showing that the NABs directed against r-IFNs do not effectively cross-react with n-IFNs.

Intranasal administration of adjuvant-combined vaccine protects monkeys from challenge with the highly pathogenic influenza A H5N1 virus.

The effectiveness in cynomolgus macaques of intranasal administration of an influenza A H5N1 pre-pandemic vaccine combined with synthetic double-stranded RNA (polyI/polyC12U) as an adjuvant was examined. The monkeys were immunized with the adjuvant-combined vaccine on weeks 0, 3, and 5, and challenged with the homologous virus 2 weeks after the third immunization. After the second immunization, the immunization induced vaccine-specific salivary IgA and serum IgG antibodies, as detected by ELISA. The serum IgG antibodies present 2 weeks after the third immunization not only had high neutralizing activity against the homologous virus, they also neutralized significantly heterologous influenza A H5N1 viruses. The vaccinated animals were protected completely from the challenge infection with the homologous virus. These results suggest that intranasal immunization with the Double stranded RNA-combined influenza A H5N1 vaccine induce mucosal IgA and serum IgG antibodies which could protect humans from homologous influenza A H5N1 viruses which have a pandemic potential.

Cross-protection against H5N1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine.

BACKGROUND: Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. METHODS: BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005-2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C(12)U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. RESULTS: Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. CONCLUSIONS: Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor-3 agonist, poly(I):poly(C(12)U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.