Patent Publication Number: US-2015064216-A1

Title: Dsrnas as influenza virus vaccine adjuvants or immuno-stimulants

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of Ser. No. 13/064,824 filed Apr. 19, 2011, which is a continuation of Ser. No. 11/634,389, filed Dec. 6, 2006, now U.S. Pat. No. 7,943,147, issued on May 17, 2011, which claims the benefits of priority to provisional application Ser. No. 60/793,239 filed Apr. 20, 2006, Ser. No. 60/752,898 filed Dec. 23, 2005 and Ser. No. 60/742,906 filed Dec. 7, 2005, the entire content of each of which is hereby incorporated by reference in this application. 
    
    
     Vaccine protection against acute or chronic viral infection is facilitated by using, together with an anti-influenza vaccine, as an adjuvant or immuno-stimulant, a dsRNA. 
     BACKGROUND OF THE INVENTION 
     Adjuvants have been used to facilitate vaccines in affording immunization to protect animals including humans. Identifying an efficient and effective adjuvant is often a difficult task. 
     Of particular interest are vaccines for protecting against influenza viruses, and of current interest avian influenza virus H5N1 (bird flu) including Vietnam and Hong Kong strains. Inactivated vaccines against influenza virus have been administered parenterally to induce serum antibodies and also to the nasal mucosa to provide mucosal immunity to influenza virus. 
     Several adjuvants are known such as alum, squalene emulsion (MF 59, Chiron Vaccines), and Freund&#39;s adjuvant. Recently a synthetic dsRNA polyriboinosinic polyribocytldylic acid or poly (I:C) has been proposed as an adjuvant or immuno-stimulant for inactivated influenza virus vaccine; see Ichinohe et al, Journal of Virology, March 2005, p. 2910-2919. 
     DESCRIPTION OF THE INVENTION 
     Disclosed are methods of facilitating vaccine protection against an acute or chronic viral infection comprising the coordinated administration to a subject requiring protection an immunity-inducing amount of an anti-influenza vaccine together with, as an adjuvant, a dsRNA. Also disclosed are methods of facilitating vaccine protection against an acute or chronic viral infection comprising administering to a subject requiring protection an immunity-inducing amount of an anti-influenza vaccine in combination with, as an adjuvant or immuno-stimulant, a dsRNA. 
     The invention includes methods of facilitating vaccine protection against an acute or chronic viral infection comprising administering substantially simultaneously or sequentially to a subject requiring protection an immunity-inducing amount of an anti-influenza vaccine together in admixture with, as an adjuvant or immuno-stimulant, a dsRNA. 
     This invention also includes methods of protecting animals, including humans, susceptible to avian influenza infections against viral-induced pathology secondary to both antigenic drift and shift (as evidenced by rearrangement of the viral particle structure) and genomic rearrangement as well. 
     The invention further includes methods of enhancing immunization against influenza viruses by coordinated administration of a vaccine to patients together or conjointly a synthetic, specifically configured, double-stranded ribonucleic acid (dsRNA). The dsRNA of choice is AMPLIGEN®, available from HEMISPHERx BIOPHARMA, 1617 JFK Boulevard, Philadelphia, Pa. USA., a synthetic, specifically configured, double-stranded ribonucleic acid (dsRNA) which retains the immunostimulatory and antiviral properties of other double-stranded RNA molecules (dsRNA) but exhibits greatly reduced toxicity. Like other dsRNAs, AMPLIGEN® can stimulate host defense mechanisms including innate immunity. AMPLIGEN® has the ability to stimulate a variety of dsRNA-dependent intracellular antiviral defense mechanisms including the 2′,5′-oligoadenylate synthetase/RNase L and protein kinase enzyme pathways. 
     In the context of the present invention, what is meant by “coordinated” use is, independently, either (i) co-administration, i.e. substantially simultaneous or sequential administration of the vaccine and of the dsRNA, or (ii) the administration of a composition comprising the vaccine and the dsRNA in combination and in a mixture, in addition to optional pharmaceutically acceptable excipients and/or vehicles. 
     The mismatched dsRNA may be of the general formula rI n ·(C 12 U) n . In this and the other formulae that follow r=ribo. Other mismatched dsRNAs for use in the present invention are based on copolynucleotides selected from poly (C m ,U) and poly (C m G) in which m is an integer having a value of from 4 to 29 and are mismatched analogs of complexes of polyriboinosinic and polyribocytidilic acids, formed by modifying rI n ·rC n  to incorporate unpaired bases (uracil or guanine) along the polyribocytidylate (rC m ) strand. Alternatively, the dsRNA may be derived from r(I)·(C) dsRNA by modifying the ribosyl backbone of polyriboinosinic acid (rI n ), e.g., by including 2′-O-methyl ribosyl residues. The mismatched may be complexed with an RNA-stabilizing polymer such as lysine and/or cellulose. Of these mismatched analogs of rI n ·rC n , the preferred ones are of the general formula rI n ·r(C 11-14 ,U) n . or rI n ·r(C 29 ,G) n , and are described by Carter and Ts&#39;o in U.S. Pat. Nos. 4,130,641 and 4,024,222, the disclosures of which are hereby incorporated by reference. The dsRNA&#39;s described therein generally are suitable for use according to the present invention. 
     Other examples of mismatched dsRNA for use in the invention include: 
       r(I)·r(C4, U)
 
       r(I)·r(C7, U)
 
       r(I)·r(C13, U)
 
       r(I)·r(C22, U)
 
       r(I)·r(C20, G)
 
       and 
       r(I)·r(C p·23 ,G &gt;p ).
 
     Alternatively the dsRNA may be the matched form, thus polyadenylic acid complexed with polyuridylic acid (poly A•poly U) may also be used. 
     Another aspect of the invention is the treatment of acute and chronic viral infections susceptible to vaccine prophylaxis therapy, available now or in the future including, for example, HIV, severe acute respiratory syndrome (SARS) and influenza including avian influenza employing a synergistic combination of an appropriate vaccine and a dsRNA. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The invention is further explained and illustrated in the following examples and figures in which: 
         FIG. 1  is a table showing the results of Example 1; 
         FIG. 2  is a table showing the results of Example 2; 
         FIG. 3  is a table showing the results of Example 3 using a trivalent influenza vaccine; 
         FIG. 4  is a table showing the results of Example 3 using a trivalent vaccine plus AMPLIGEN® intranasally; 
         FIG. 5  is a table showing a direct cross assessment according to Example 3 of trivalent seasonal influenza vaccine and intranasally administered AMPLIGEN®; and 
         FIG. 6  is a table showing the results of Example 3. 
     
    
    
     The terms used in the Figures that follow are: 
     A/VN avian influenza/Vietnam (H5N1) strain 
     VN1194 avian influenza/Vietnam (H5N1) strain 
     05/06 Vaccine trivalent “seasonal” influenza vaccine for the 2005-2006 season 
     Amp AMPLIGEN® 
     I.N. intranasal 
     S.C. subcutaneous 
     Anti-A/VN IgA IgA antibodies raised against the avian influenza Vietnam strain 
     Anti-A/VN IgG IgG antibodies raised against the avian influenza Vietnam strain 
     A/VN virus titer quantitation of the amount of avian influenza virus Vietnam strain (i.e. as detected in nasal mucosal washings) 
     Anti-05/06 Vaccine antibodies raised against the 2005/2006 trivalent seasonal influenza vaccine 
     H5N1 avian influenza virus classification type 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Example 1 
     Cross Protection Between Avian Influenza Strains 
     This study was conducted in mice in the manner of Ichinohe et al, Journal of Virology, March, 2005, pages 2910-2919, this time using two different strains of avian flu virus, Vietnam and Hong Kong, and the dsRNA AMPLIGEN®, as described above, in combination or alone with the vaccine. The results are given in  FIGS. 1 and 2 . 
     In the first panel, from the antibodies detected in the nasal wash use of the (A/VN) vaccine by itself when administered intranasally provided a positive result in raising antibody but when administered with AMPLIGEN® produced a result that was more than twice than that of the vaccine used alone. No IgA antibodies were detected using AMPLIGEN® alone. The subcutaneous route did not yield any IgA antibodies in the nasal mucosa. 
     In contrast to this, a limited number of IgG antibodies were raised in the blood serum following intranasal administration but significantly greater amounts were obtained in the blood serum from the subcutaneous administration. Again, the combination of the vaccine plus AMPLIGEN® produced a greater result than with the vaccine alone. 
     The animals were then subjected to a challenge to avian influenza virus Vietnamese strain and, significantly, there was no virus detected in the nasal wash of the challenged animals receiving a combination of vaccine and AMPLIGEN® administered by the intranasal route while various amounts of virus were detected using the vaccine alone, AMPLIGEN® alone, intranasally, and a combination of vaccine and AMPLIGEN® administered subcutaneously. 
     It is desirable to raise antibodies to the avian flu virus in the nasal mucosa and other mucosa as this is the typical point of entry/infection and is believed to offer a significant preventative or mitigating benefit. 
     Example 2 
     Cross Protection Between Seasonal Influenza Vaccine and H5N1 
     A second set of studies was completed similar to Example 1, this time initially using inactivated avian influenza virus vaccine Vietnam strain in combination with AMPLIGEN® or AMPLIGEN® alone or the vaccine alone then later challenging with the different Hong Kong strain of avian influenza virus. The results are shown in  FIG. 2 . The first two panels under anti-A/VN-IgA and anti-A/VN IgG were prior to challenge and the third panel was subsequent to challenge with the Hong Kong strain. Overall, beneficial results were noted in the virus titer nasal wash subsequent to challenge with the best results achieved using the combination of Vietnam strain vaccine and AMPLIGEN® and subsequent challenge with the Hong Kong strain of the virus. 
     These results indicate continued efficacy when the Vietnam strain vaccine-treated patients also receiving AMPLIGEN® were later challenged with the Hong Kong strain of the virus and from this it is expected that similar results will occur when the viral strains are reversed and the Hong Kong virus is used to raise the vaccine followed by subsequent challenge with the Vietnam strain. 
     Example 3 
     Viral Antigen Sparing and Augmentation 
     In this example a study was made to determine how the influence of poly(I:C) on the administration of an avian influenza, Vietnam strain in animals similar to those used in Example 2. The results are presented in  FIG. 6 . Various doses of the avian influenza vaccine (A/VN) were employed and varying amounts of poly(I:C) were used including no A/VN and no poly(I:C) as controls. Of particular interest is a comparison between 1 μg of avian influenza vaccine and no poly(I:C) contrasted with 0.1 μg of avian influenza vaccine and 10 μg of poly(I:C). When administered intranasally in the first panel of bar graphs it will be noted that more antibodies were raised by the combination of 0.1 μg of A/VN and 10 μg of poly(I:C) compared to a tenfold larger amount of avian influenza vaccine used by itself. Of particular significance is the final panel under the heading Survival Rate where the survival rate was numerically the same, on a percentage basis, between the use of one-tenth the amount of avian influenza vaccine in combination with 10 μg poly(I:C) and 10 μg of A/VN alone (without poly(I:C)). Note also the A/VN virus titer in the nasal wash was rather insignificant for the combination of 0.1 μg A/VN and 10 μg poly(I:C) as compared to a measurable value when the avian flu vaccine was used alone. From these data one may conclude the use of poly(I:C) as an adjuvant enables one to reduce by tenfold (in this example) the amount of avian influenza vaccine necessary to achieve significant rates of survival. 
     Presence of the AMPLIGEN® appears to possess cross-protection ability against variant avian influenza viruses and thereby mitigate antigenic drift of the avian influenza virus. 
     Antigenic drift is a change in structure of a virus, such as the internal and external proteins, glycoproteins, glycolipids, etc., due to fundamental change in the genomic content of the virus particle. dsRNAs reduce the phenomenon of viral escape and cellular damage attendant thereto. Viral escape is a process by which a virus or intracellular pathogen alters its host range or indirectly alters its susceptibility of antiviral or immunological therapies. 
     This invention includes methods of cross-protecting animals, including humans, susceptible to avian influenza infections against viral-induced pathology secondary to both antigenic drift and shift (produced by mutations or rearrangement of the viral genetic material). 
     In  FIG. 3 , seven groups of mice, five mice per group, were selected. Four of these groups were exposed to the 2005/2006 trivalent influenza vaccine either intranasally or subcutaneously. Within 21 days intranasal inoculation was repeated and within 14 days intranasal inoculation was completed again making a total of one initial inoculation and two boosters. 
     Two weeks after the second booster the mice were then subjected to challenge with the avian influenza VN1194 (H5N1) strain and assessed for the presence and amount of IgA anti-A/VN in a nasal wash and for IgG antibodies in serum. The results indicate that only with the presence AMPLIGEN® and administration by the intranasal route were A/VN IgA antibodies raised against the avian influenza Vietnam (VN1194) strain. While IgG antibodies were raised in the serum against the VN1194 strain from the intranasal administration there were serum antibodies raised with or without the presence of AMPLIGEN® using the SC route of administration. Virus titers for the avian flu virus were then assessed after avian influenza VN1194 (H5N1) virus challenge in nasal wash. For the subset receiving both the trivalent seasonal vaccine and AMPLIGEN® adjuvant the virus was effectively neutralized while the other groups showed measurable quantities of the A/VN virus. 
       FIG. 4  shows that the only group of animals to survive the challenge with VN1194 as assessed over a period of 18 days, was the group which received both the 05/06 trivalent vaccine plus the AMPLIGEN® intranasally. While antibodies were present in the blood serum they provided no effective protection against VN1194 challenge but antibodies present in the nasal mucosa were effective to prevent infection and death over the period of time measured. These findings are significant as they demonstrate in this study protection against avian influenza H5N1 strains is conferred by the use of a trivalent seasonal vaccine administered intranasally with AMPLIGEN® as a vaccine adjuvant. 
       FIG. 5  shows the direct cross assessment, again indicating the quantities and amounts of 05/06 trivalent vaccine, AMPLIGEN® and route of administration but measuring for the antibodies to be elicited against the seasonal trivalent vaccine as measured either in the nasal mucosa or blood serum. The results show antibodies against the seasonal vaccine were present in the nasal mucosa of only those animals receiving both the trivalent 05/06 seasonal vaccine and AMPLIGEN® administered by the intranasal route. Regarding the detecting of antibodies against the 05/06 trivalent vaccine in serum, all of the groups had a certain elevated “baseline” level, but a significant increase was seen both times the vaccine was used with AMPLIGEN®. 
     Our studies also demonstrate the presence of antibodies in blood serum does not necessarily provide an accurate indicator of protection against avian influenza and the more reliable indicator is the antibodies raised in the nasal mucosa. 
     
       
         
           
               
            
               
                   
               
               
                 Additional key cellular mechanisms induced by double- 
               
               
                 stranded RNAs to provide for more potent  
               
               
                 immune stimulating effects of influenza and other vaccines. 
               
            
           
           
               
               
               
            
               
                 Target 
                 Activity 
                 Result 
               
               
                   
               
               
                 Epithelial cells 
                 Activate antiviral defenses 
                 Restricts viral replication 
               
               
                   
                 Secrete interferon. 
                 in infected, and 
               
               
                   
                   
                 surrounding cells. 
               
               
                   
                   
                 Initiate supportive immune 
               
               
                   
                   
                 response. 
               
               
                 Dendritic Cells 
                 Activate DC antigen 
                 T cell activation and 
               
               
                   
                 presentation, costimulatory 
                 differentiation into T 
               
               
                   
                 function, and inflammatory 
                 helper cells, and T killer 
               
               
                   
                 cytokine release. 
                 CTL cells. 
               
               
                 Macrophages 
                 Activate phagocytosis and 
                 Increased killing and 
               
               
                   
                 inflammatory cytokine 
                 clearing of virally infected 
               
               
                   
                 release. 
                 cells. 
               
               
                 Mast cells 
                 Cytokine release 
                 Enhance recruitment and 
               
               
                   
                   
                 activation of immune cells 
               
               
                   
                   
                 at affected tissue sites. 
               
               
                 Natural Killer  
                 Lysis of virally infected 
                 Enhance viral clearance 
               
               
                 (NK) cells 
                 cells, Further dendritic cell 
                 and boost immune 
               
               
                   
                 activation. 
                 responses. 
               
               
                 Gamma-delta  
                 Activate innate sentinel T 
                 Enhance immune 
               
               
                 T cells 
                 cells in epithelial tissues. 
                 responses. 
               
               
                 CD4 and CD8  
                 Augment T cell activation, 
                 Enhance magnitude of 
               
               
                 T cells 
                 differentiation, cytokine 
                 immune responses. 
               
               
                   
                 secretion, and survival 
               
               
                   
               
            
           
         
       
     
     Avian influenza co-administration studies were extended to a primate model, where vaccination plus co-administered AMPLIGEN® was well tolerated and effective. In this study macaques were vaccinated with A/VN plus AMPLIGEN® (A/Vietnam (H5N1) 90 μg/500 ml, AMPLIGEN® 500 μg), for three doses, spaced 3 and 2 weeks apart. That is, an initial dose, 3 weeks later a second dose and 2 weeks later a third dose. Then the monkeys were challenged 2 weeks after the third does with high doses of A/VN (A/Vietnam (H5N1) 2.5×10 5  pfu/2.5 ml (lung) and A/Vietnam (H5N1) 0.5×10 5  pfu/0.5 ml (nasal)) intra-tracheally and intranasally. Infected control animals developed tachypnea, coughing, weight loss, and focal consolidating pneumonia. Vaccinated animals were symptom free, and protected from disease with normal appearing pulmonary tissue. 
     While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.