Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge.

The avian H5N1 influenza virus has the potential to cause a new pandemic. Since it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to produce vaccines that confer cross-protective immunity. Mucosal vaccine administration was reported to induce cross-protective immunity by inducing secretion of IgA at the mucosal surface. Adjuvants can also enhance the development of fully protective mucosal immunity. Here we show that a new mucosal adjuvant, poly I:poly C12U (Ampligen), a Toll-like receptor 3 agonist proven to be safe in a Phase III human trial, is an effective adjuvant for H5N1 influenza vaccination. Intranasal administration of a candidate influenza vaccine with Ampligen resulted in secretion of IgA, and protected mice that were subsequently challenged with homologous A/Vietnam/1194/2004 and heterologous A/HK/483/97 and A/Indonesia/6/2005 virus.

Immunological responses after immunisation of mice with microparticles containing antigen and single stranded RNA (polyuridylic acid).

Certain toll-like receptor (TLR) agonists, e.g. CpG DNA, can be used as potent vaccine 'adjuvants'. It is known that some sequences of single stranded (ss) RNA stimulate proinflammatory and antiviral responses following interaction with TLR 7 and 8. We have encapsulated ovalbumin (OVA) in the presence and absence of polyuridylic acid (poly-U) inside polylactide microparticles. In comparison to microparticles containing only OVA, bulk cultures of bone marrow-derived plasmacytoid and myeloid dendritic cells produced more (P<0.05) IL-12 and interferon (IFN)-alpha when stimulated with microparticles containing OVA and poly-U. Subcutaneous injection of comicroencapsulated OVA and poly-U resulted in statistically elevated levels of serum anti-OVA IgG1 (P<0.05 versus naïve mice). Conversely, anti-OVA IgG1 levels in C57 BL6 mice immunised with OVA loaded microparticles (without RNA) were statistically indifferent to naïve animals. Furthermore, injection of coencapsulated OVA and poly-U resulted in (P<0.05) greater numbers of OVA specific IFN-gamma secreting T-cells as compared with mice injected with OVA loaded microparticles. A similar trend was seen in mice immunised with OVA loaded microparticles decorated with CpG or solutions of admixed OVA and CpG (P<0.05). These data demonstrate, for the first time, that appropriately formulated ssRNA can act as a potent adjuvant and modulator of adaptive immunological responses.

Effect of interferon-alpha and interferon-inducers on West Nile virus in mouse and hamster animal models.

The recent West Nile virus (WNV) outbreak in the United States has increased the need to identify effective therapies. Studies were conducted in cell culture and in rodent animal models to determine the efficacy of interferon-alpha (IFN-alpha), interferon (IFN) inducers and ribavirin, alone or in combination with IFN, in treating WNV. Intraperitoneal injection of IFN-alpha B/D (qd for 7 days), polyI-polyC(12)U [Ampligen (every other day for 7 days)] and topically applied imiquimod (qd for 7 days), administered from 1 day before viral challenge, were effective in protecting, respectively, 100%, 100% and 70% of BALB/c mice from mortality induced by subcutaneous injection of WNV. When IFN-alpha B/D or Ampligen treatments were delayed to 4-6 h before viral challenge in mice, efficacy was greatly diminished. Infected Syrian golden hamsters treated with interferon alphacon-1 (Infergen) and Ampligen 4-6 h before viral challenge gained more weight and had a greater survival than saline-treated animals. A combination study of subcutaneously administered Infergen (5 to 0.05 microg/kg/day) and ribavirin (75 to 7.5 mg/kg/day) in >7 week old hamsters demonstrated that Infergen was slightly efficacious in reducing mortality and disease signs; however, it was not synergistic in its antiviral effects when combined with ribavirin. Ribavirin treatment alone increased mortality of infected hamsters. The reduced mortality correlated with reduced plasma viraemia. Since WNV-infected patients have already been treated with IFN and ribavirin and future clinical trials have been suggested, this first report of IFN alone or in combination with ribavirin in WNV-infected animal models might provide useful information for subsequent treatment of patients.

New "ThetaSwitch" drug may increase long-term survival in HIV disease.

AIDS: Hemisphere Biopharma reports that its nucleic acid compound, Ampligen, in conjunction with ZDV, switched a significant percentage of HIV-infected clinical trial subjects from an unresponsive Theta status to a responsive one. Driving the immune reaction in the apparently correct direction by selectively controlling the Theta Switch was demonstrated in vivo by the ability of certain nucleic acids to throw the Switch correctly and to elicit the production of a specific biological cocktail of cytokines, including interferon, interleukin-2, and interleukin-12. These mediators or biochemical workhorses of the Theta 1 Switch apply to the cell-mediated immune response, or positive skin test response.^ieng

In vivo antiviral effects of mismatched double-stranded RNA on duck hepatitis B virus.

The antiviral activity and ability of mismatched double-stranded RNA (m-dsRNA), r(I)n.r(C12-U)n, to induce interferon (IFN) were evaluated in ducks chronically infected with duck hepatitis B virus (DHBV). When m-dsRNA was administered intravenously at a single dose of 5 mg/kg, serum DHBV DNA concentrations decreased significantly for 3 days (P < 0.002). However, the DHBV DNA concentrations returned to the pretreatment levels 4 days after treatment. Inhibition of DHBV DNA replication in the liver was also observed 2 days after treatment. Serum IFN activity peaked 3 hours after administration of m-dsRNA, then rapidly declined. 2'-5' Oligo-adenylate synthetase (2'-5'AS) activity increased gradually after treatment and remained elevated for at least 48 hours. In ducks receiving m-dsRNA once daily for 7 consecutive days, serum DHBV DNA concentrations on the last day of treatment were decreased by 76 +/- 12% (P < 0.05) in ducks that received 0.2 mg of m-dsRNA per kg and by 65 +/- 12% (P < 0.05) in ducks that received 1 mg of m-dsRNA per kg. This decrease persisted for at least 2 weeks after the cessation of treatment in all ducks. These results suggest that m-dsRNA effectively inhibits DHBV replication in vivo, and that IFN induction and stimulation of 2'-5'AS activity contribute to the inhibition of DHBV replication by m-dsRNA.

The use of ampligen alone and in combination with ganciclovir and coumermycin A1 for the treatment of ducks congenitally-infected with duck hepatitis B virus.

Ampligen, a known immunomodulator and interferon inducer, was used alone and in combination with other antiviral agents to treat ducks congenitally-infected with duck hepatitis B virus. These antiviral agents included the conventional nucleoside analogue ganciclovir and the prokaryotic DNA gyrase B inhibitor coumermycin A1. When used alone, ampligen decreased the amount of serum and liver viral DNA, but had no effect on circulating duck hepatitis B surface antigen (DHBsAg). In combination with ganciclovir, the antiviral effect appeared at least additive with a greater inhibition of viral DNA replication within the liver. The combination of ampligen with coumermycin A1 also resulted in inhibition of viral replication but to a lesser extent than ampligen alone. When all three agents were used together, viral DNA replication was again inhibited, but as with previous treatment regimes, serum DHBsAg levels remained unchanged. At the end of the treatment period for all regimes, analysis of viral DNA forms in the liver showed that the viral relaxed circular and supercoiled DNA forms had persisted. Within 1 week of cessation of therapy, viral replication had often returned to pre-treatment levels. Interferon-like activity was detected in the sera of the majority of the treated ducks during the ampligen therapy, but no clear relationship between the presence of interferon and antiviral effect could be established. These observations in the duck hepatitis B model may provide a rational basis for the use of combinations of antiviral and immunomodulatory regimes for the management of chronic hepatitis B infection in man.

Biologic effects after a single dose of poly(I):poly(C12U) in healthy volunteers.

Poly(I):poly(C12U) (mismatched double-stranded RNA; atvogen), an interferon inducer, is active against human immunodeficiency virus in vitro. To determine the extent and duration of the biologic effects of poly(I):poly(C12U), we administered a single dose of the drug to healthy volunteers in a randomized, double-blind, placebo-controlled 2-week crossover study. We analyzed blood for alpha and gamma interferons, neopterin, 2',5'-oligoadenylate synthetase, lymphocyte surface markers, lymphocyte proliferation after exposure to soluble antigens and mitogens, and natural killer cell activity. Minimal biologic effects were observed after administration of a single 200-mg dose to four volunteers; therefore, the dose was increased to 600 mg in 10 subjects. Only neopterin levels and symptoms were greater after administration of 600 mg of poly(I):poly(C12U) than after administration of placebo (Wilcoxon signed rank sum test, P = 0.06). A definite response in 2',5'-oligoadenylate synthetase activity, however, was seen in a few subjects. Neither alpha nor gamma interferon was detectable in serum after poly(I):poly(C12U) dosing. The neopterin changes after administration of poly(I):poly(C12U) were similar at both poly(I):poly(C12U) dose levels, with an early decrease at 6 h, a peak at 1 day, and a gradual decrease toward the baseline over the following 3 days. A mild flu-like syndrome occurred in one-half of the subjects following administration of poly(I):poly(C12U) and in only one subject following administration of placebo. This syndrome resolved within 16 h after poly(I):poly(C12U) dosing. We conclude that poly(I):poly(C12U) does not induce measurable levels of interferon and causes only minimal biologic or toxic effects among those parameters measured after administration of a single dose in the 200- to 600-mg dose range in health volunteers.

A phase I study of ampligen in human immunodeficiency virus-infected subjects.

Ampligen, poly(I)n:poly(C12U)n, was administered intravenously to 39 human immunodeficiency virus (HIV)-infected subjects, asymptomatic or with early AIDS-related complex (ARC) and with CD4+ cell counts less than 500/mm3 in a phase I dose-escalation study. Six doses ranging from 10 to 570 mg/m2 were administered twice-weekly for 9-25 weeks to groups of 5-7 subjects. There was no significant effect on HIV as measured by serum p24 levels, the proportion of patients from whom HIV could be cocultured from blood, or the concentration of peripheral mononuclear cells positive for the virus. Although patients on 10 and 40 mg/m2 showed a significant decline in CD4+ cell counts, as would be expected in untreated patients, patients who received doses greater than or equal to 120 mg/m2 showed no significant decline in CD4+ cell counts. In addition, there was a significant increase in CD4+ cell counts with respect to dose of ampligen. This effect of ampligen and the fact that it has been shown to act synergistically with zidovudine against HIV in vitro suggest that the combination might be tried clinically in patients.

An hypothesis concerning optimal therapy in HIV disease.

Ampligen, a mismatched double stranded RNA, is hypothesized to be an ideal base therapy for HIV disease to which other agents, such as the nucleoside analogue, AZT, can be advantageously added. The unique properties of Ampligen which support this hypothesis include activation of immune cells, inhibition of virus replication by inducing an antiviral cellular state and inhibition of growth of neoplastic cells. Ampligen is synergistic with other agents being used or being tested for use in HIV disease and is without toxicity.

Continued studies on the adjuvancy effect of natural and synthetic double-stranded RNA preparations with inactivated Newcastle disease vaccines in fowls.

The adjuvant effect of the natural ds-RNA, BRL 5907, with inactivated Newcastle disease vaccines was confirmed using different oil-based formulations and could be given either as single injections or separately at adjacent sites. A minimum dose level of 0-04 mg BRL 5907 per bird was required for a significant enhancement of antibody levels following vaccination. While the synthetic ds-RNA Poly IC gave a similar response to that observed with BRL 5907, no significant effects could be detected with two other natural ds-RNA materials.

Studies on hypoxia: XII. Detrimental effects of synthetic polyribonucleotides on epiphyseal plates of rats exposed to hypoxia.

The effect of different doses of polyadenylic and polyuridylic acids (poly A:U) was studied in control rats and in rats exposed to hypoxia. In the control rats, administration of different doses of poly A:U did not change the thickness of the epiphyseal plate or increase the incorporation of 3H-phenylalanine as judged using radioautography. Rats exposed to hypoxia showed a significant dose-related reduction in the thickness of the epiphyseal plate and 3H-phenylalanine incorporation.