David R. Strayer

Cross-Protection against H5N1 Influenza Virus Infection Is Afforded by Intranasal Inoculation with Seasonal Trivalent Inactivated Influenza Vaccine

Abstract 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(C12U) (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(C12U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.

Familial occurrence of breast cancer is associated with reduced natural killer cytotoxicity

SummaryA factor in the incidence of spontaneous neoplasms in mice is the endogenous level of natural cell-mediated cytotoxicity (1, 2). These immunosurveillant or host defense mechanisms are probably under the control of multiple gene products (3, 4) including interferons. We studied natural killer (NK) cytotoxicity using peripheral blood mononuclear cells from 59 normal individuals with either a high (17) or low (42) familial incidence of breast cancer. The K562 cell line was used as target in51Cr release assays. Three effector: target ratios (6.2 : 1, 25 : 1, and 50 : 1) were studied in quadruplicate using 3, 4 and 5-h incubations. Significantly lower natural killer activity (p<0.002) was detected in normal individuals with high familial incidences of breast cancer compared to individuals with low incidences in each of the three separate assays (50 : 1). The same conclusion was reached whether the data were expressed in terms of lytic units per 107 blood mononuclear cells or as % specific51Cr released. Thus, a relationship was observed between the occurrence of breast cancer in closely related family members and low natural cell-mediated cytotoxicity. This result suggests that defects in NK activity may play a role in the initiation of human breast tumors. However, prospective studies will be necessary to establish whether low NK cell activity is a risk factor for breast cancer.

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: C12U), 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

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. J. Med. Virol. 82:1754–1761, 2010.

Mismatched double-stranded RNA, ampligen (poly(I) : poly(C12U)), demonstrates antiviral and immunostimulatory activities in HIV disease

Mismatched double-stranded RNA (Ampligen) has broad spectrum antiviral and immunomodulatory activities. These activities generate stabilization or improvement in three important surrogate markers of HIV disease progression. Patients with HIV disease treated with Ampligen do not become positive for p24 antigen, in contrast to patients treated with AZT or placebo. Viral burden can also be decreased in patients receiving Ampligen/AZT therapy. In vitro studies indicate that both AZT sensitive and AZT resistant viruses can be inhibited by Ampligen alone and are synergistically inhibited by Ampligen in combination with AZT. The immunomodulatory effects of Ampligen are manifested as a stabilization of CD4 counts. When Ampligen is combined with AZT, an increase in CD4 count is seen. Furthermore, a return or increase in delayed type hypersensitivity to mumps, Candida, and trichophyton was seen in approximately 70% of patients treated with Ampligen. The activity of Ampligen in HIV disease is due to its multifunctional activity as an antiviral and immune stimulating agent. The antiviral effect directly inhibits HIV-infection and other viruses which have been implicated in HIV disease acceleration and progression. The immunomodulatory activity can stabilize, increase, or restore immune function. This enhanced immune function can also lead to the further inhibition of additional infections associated with disease progression. Thus, Ampligen has multiple mechanisms of action against HIV disease.

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, β-1a, and β-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.

Divergence of primate ribosomal RNA genes as assayed by restriction enzyme analysis

Primate ribosomal RNA (rRNA) genes have been compared by restriction endonuclease mapping. In all species examined, the restriction map of the reiterated ribosomal DNA is simple (within the limits of detection by hybridization with rRNA) and is consistent with a high degree of homogeneity among the repeats. Within a species, all members have similar rDNA restriction patterns. However, different species of primates have distinctly different rDNA restriction maps; even chimpanzee and man can be discerned by their rDNA restriction patterns. Possible mechanisms for maintenance of homogeneity of the rDNA repeats within a species, while allowing divergence among closely related species, are discussed.

Quantitation of RNA-dependent platelet DNA polymerase in patients with myeloproliferative disorders.

Platelets from 28 patients with the myeloproliferative diseases (MPD) polycythaemia vera (9), essential thrombocythaemia (6), myelofibrosis with myeloid metaplasia (5) and chronic myelogenous leukaemia (8) were examined for an RNA‐dependent DNA polymerase activity using standardized conditions permitting highly reproducible quantitation. Low levels of activity were detected in platelets of normal individuals, but platelets of nearly all MPD patients (25/28) possessed higher levels. The polymerase activity correlated with diagnosis (P= 0.001) and did not correlate with platelet counts (P>0.2). Quantitation of this RNA‐dependent DNA polymerase activity may be a useful parameter in the diagnosis of myeloproliferative disorders.

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.

Antitumor effects of interleukin-2 and mismatched double-stranded RNA, individually and in combination, against a human malignant melanoma xenograft

SummaryThe antitumor effects of recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA) were assessed in tissue culture and in a nude mouse model. Mismatched dsRNA did not show a direct antiproliferative effect against the human malignant melanoma cell line, BRO, in tissue culture. However, treatment of the BRO cells with up to 1000 units/ml rIL-2 in culture showed a slight increase in growth rate. Combined rIL-2/mismatched dsRNA treatment also demonstrated a similar slight enhancement of growth. Nude mice bearing subcutaneous tumors were treated by intraperitoneal injection of low doses (5000–20 000 units) of rIL-2 and mismatched dsRNA (500 µg). The in vivo tumor growth was significantly inhibited by the combined treatments (P <0.05) and survival was significantly increased (P <0.05). Measurement of cytotoxicity using splenocytes from treated animals showed significant augmentation of lytic activity against natural killer(NK)-sensitive YAC-1 cells in all rIL-2/mismatched dsRNA treatment groups, compared to the individual treatments or controls (P <0.05). Cytotoxicity of the splenocytes against the NK-resistant BRO cells was also augmented in animals treated with mismatched dsRNA and the highest rIL-2 dose utilized here (P <0.01). Renal, liver, and hematological toxicity was evaluated by measurement of blood urea nitrogen, creatinine, serum asparrtate aminotransferase, and a complete blood count with differential. There were no significant differences in these parameters in any of the treatment groups. Similarly, no differences in weight of the animals was seen in any treatment group. These results indicate that the combination of low-dose rIL-2 and mismatched dsRNA can potentiate host-mediated antitumor effects, yielding increased survival, without significant toxicity.