News Column

Researchers Submit Patent Application, "Vaccine against Rsv", for Approval

June 19, 2014



By a News Reporter-Staff News Editor at Gene Therapy Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors RADOSEVIC, KATARINA (LEIDEN, NL); CUSTERS, JEROME H. H. V. (ALPHEN AAN DEN RIJN, NL); VELLINGA, JORT (LEIDEN, NL); WIDJOJOATMODJO, MYRA N. (LEIDEN, NL), filed on March 22, 2013, was made available online on June 5, 2014 (see also Crucell Holland B.v.).

The patent's assignee is Crucell Holland B.v.

News editors obtained the following quote from the background information supplied by the inventors: "After discovery of RSV in the 1950s, the virus soon became a recognized pathogen associated with lower and upper respiratory tract infections in humans. Worldwide, it is estimated that 64 million RSV infections occur each year resulting in 160.000 deaths (WHO Acute Respiratory Infections Update September 2009). The most severe disease occurs particularly in premature infants, the elderly and immunocompromised individuals. In children younger than 2 years, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of the hospitalizations due to respiratory infections, and the peak of hospitalization occurs at 2-4 months of age. It has been reported that almost all children have been infected by RSV by the age of two. Repeated infection during lifetime is attributed to ineffective natural immunity. The level of RSV disease burden, mortality and morbidity in the elderly are second to those caused by nonpandemic influenza A infections.

"RSV is a paramyxovirus, belonging to the subfamily of pneumovirinae. Its genome encodes for various proteins, including the membrane proteins known as RSV Glycoprotein (G) and RSV fusion (F) protein which are the major antigenic targets for neutralizing antibodies. Proteolytic cleavage of the fusion protein precursor (F0) yields two polypeptides F1 and F2 linked via disulfide bridge. Antibodies against the fusion-mediating part of the F1 protein can prevent virus uptake in the cell and thus have a neutralizing effect. Besides being a target for neutralizing antibodies, RSV F contains cytotoxic T cell epitopes (Pemberton et al., 1987, J. Gen. Virol. 68: 2177-2182).

"Treatment options for RSV infection include a monoclonal antibody against the F protein of RSV. The high costs associated with such monoclonal antibodies and the requirement for administration in a hospital setting, preclude their use for prophylaxis in the at-risk population at large scale. Thus, there is a need for an RSV vaccine, which preferably can be used for the pediatric population as well as for the elderly.

"Despite 50 years of research, there is still no licensed vaccine against RSV. One major obstacle to the vaccine development is the legacy of vaccine-enhanced disease in a clinical trial in the 1960s with a formalin-inactivated (FI) RSV vaccine. FI-RSV vaccinated children were not protected against natural infection and infected children experienced more severe illness than non-vaccinated children, including two deaths. This phenomenon is referred to as 'enhanced disease.'

"Since the trial with the FI-RSV vaccine, various approaches to generate an RSV vaccine have been pursued. Attempts include classical live attenuated cold passaged or temperature sensitive mutant strains of RSV, (chimeric) protein subunit vaccines, peptide vaccines and RSV proteins expressed from recombinant viral vectors. Although some of these vaccines showed promising pre-clinical data, no vaccine has been licensed for human use due to safety concerns or lack of efficacy.

"Adenovirus vectors are used for the preparation of vaccines for a variety of diseases, including disease associated with RSV infections. The following paragraphs provide examples of adenovirus-based RSV candidate vaccines that have been described.

"In one approach, RSV.F has been inserted into the non-essential E3 region of replication competent adenovirus types 4, 5, and 7. Immunization in cotton rats, intranasal (i.n.) application of 10.sup.7 pfu, was moderately immunogenic, and protective against lower respiratory tracts against RSV challenge, but not protective against upper respiratory tract RSV challenge (Connors et al., 1992, Vaccine 10: 475-484; Collins, P. L., Prince, G. A., Camargo, E., Purcell, R. H., Chanock, R. M. and Murphy, B. R. Evaluation of the protective efficacy of recombinant vaccinia viruses and adenoviruses that express respiratory syncytial virus glycoproteins. In: Vaccines 90: Modern Approaches to New Vaccines including prevention of AIDS (Eds. Brown, F., Chanock, R. M., Ginsberg, H. and Lerner, R. A.) Cold Spring Harbor Laboratory, New York, 1990, pp 79-84). Subsequent oral immunization of a chimpanzee was poorly immunogenic (Hsu et al., 1992, J Infect Dis. 66:769-775).

"In other studies (Shao et al., 2009, Vaccine 27: 5460-71; U.S. 2011/0014220), two recombinant replication incompetent adenovirus 5 vectors carrying nucleic acid encoding the transmembrane truncated (rAd-F0.DELTA.TM) or full-length (rAd-F0) version of the F protein of the RSV-B1 strain were engineered and given via the intranasal route to BALB/c mice. Animals were primed i.n. with 10.sup.7 pfu and boosted 28 days later with the same dose i.n. Although the anti-RSV-B1 antibodies were neutralizing and cross-reacting with RSV-Long and RSV-A2 strain, immunization with these vectors protected only partially against RSV B1 challenge replication. The (partial) protection with rAd-F0.DELTA.TM was slightly higher than with rAd-F0.

"In another study, it was observed that BALB/c mice i.n. immunization with 10.sup.11 virus particles with the replication deficient (Ad5 based) FG-Ad adenovirus expressing wild type RSV F (FG-Ad-F) reduced lung viral titers only a 1.5 log 10 compared with the control group (Fu et al., 2009, Biochem. Biophys. Res. Commun. 381: 528-532.

"In yet other studies, it was observed that intranasally applied recombinant Ad5-based replication-deficient adenovector expressing codon optimized soluble F1 fragment of F protein of RSV A2 (amino acid 155-524) (10.sup.8 PFU) could reduce RSV challenge replication in the lungs of BALB/c mice compared to control mice, but mice immunized by the intramuscular (i.m.) route did not exhibit any protection from the challenge (Kim et al., 2010, Vaccine 28: 3801-3808).

"In other studies, adenovectors Ad5-based carrying the codon optimized full-length RSV F (AdV-F) or the soluble form of the RSF F gene (AdV-Fsol) were used to immunize BALB/c mice twice with a dose of 1.times.10.sup.10 OPU (optical particle units: a dose of 1.times.10.sup.10 OPU corresponds with 2.times.10.sup.8 GTU (gene transduction unit)). These vectors strongly reduced viral loads in the lungs after i.n. immunization, but only partially after subcutaneous (s.c.) or i.m. application (Kohlmann et al., 2009, J Virol 83: 12601-12610; U.S. 2010/0111989).

"In yet other studies, it was observed that intramuscular applied recombinant Ad5-based replication-deficient adenovector expressing the sequenced F protein cDNA of RSV A2 strain (10.sup.10 particle units) could reduce RSV challenge replication only partially in the lungs of BALB/c mice compared to control mice (Krause et al., 2011, Virology Journal 8:375-386)

"Apart from not being fully effective in many cases, the RSV vaccines under clinical evaluation for pediatric use and most of the vaccines under pre-clinical evaluation, are intranasal vaccines. The most important advantages of the intranasal strategy are the direct stimulation of local respiratory tract immunity and the lack of associated disease enhancement. Indeed, generally the efficacy of, for instance, the adenovirus based RSV candidate vaccines appears better for intranasal administration as compared to intramuscular administration. However, intranasal vaccination also gives rise to safety concerns in infants younger than 6 months. Most common adverse reactions of intranasal vaccines are runny nose or nasal congestion in all ages. Newborn infants are obligate nasal breathers and thus must breathe through the nose. Therefore, nasal congestion in an infant's first few months of life can interfere with nursing, and in rare cases can cause serious breathing problems.

"More than 50 different human adenovirus serotypes have been identified. Of these, adenovirus serotype 5 (Ad5) has historically been studied most extensively for use as gene carrier. Recombinant adenoviral vectors of different serotypes may however give rise to different results with respect to induction of immune responses and protection. For instance, WO 2012/021730 describes that simian adenoviral vector serotype 7 and human adenoviral vector serotype 5 encoding F protein provide better protection against RSV than a human adenoviral vector of serotype 28. In addition, differential immunogenicity was observed for vectors based on human or non-human adenovirus serotypes (Abbink et al., 2007, J Virol 81: 4654-4663; Colloca et al., 2012, Sci Transl Med 4, 115ra2). Abbink et al., conclude that all rare serotype human rAd vectors studied were less potent than rAd5 vectors in the absence of anti-Ad5 immunity. Further it has been recently described that, while rAd5 with an Ebolavirus (EBOV) glycoprotein (gp) transgene protected 100% of non-human primates, rAd35 and rAd26 with EBOV gp transgene provided only partial protection and a heterologous prime-boost strategy was required with these vectors to obtain full protection against ebola virus challenge (Geisbert et al., 2011, J Virol 85: 4222-4233). Thus, it is a priori not possible to predict the efficacy of a recombinant adenoviral vaccine, based solely on data from another adenovirus serotype.

"Moreover, for RSV vaccines, experiments in appropriate disease models such as cotton rat are required to determine if a vaccine candidate is efficacious enough to prevent replication of RSV in the nasal tract and lungs and at the same time is safe, i.e., does not lead to enhanced disease. Preferably such candidate vaccines should be highly efficacious in such models, even upon intramuscular administration."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "It was surprisingly found by the present inventors that recombinant adenoviruses of serotype 26 (Ad26) that comprise a nucleotide sequence encoding RSV F protein are very effective vaccines against RSV in a well established cotton rat model, and have improved efficacy as compared to data described earlier for Ad5 encoding RSV F. It is demonstrated that even a single administration, even intramuscularly, of Ad26 encoding RSV F is sufficient to provide complete protection against challenge RSV replication.

"The vaccines hereof based on Ad26 surprisingly appear more potent than the ones described in the prior art that were based upon Ad5, since the Ad5-based vaccines failed to provide complete protection against RSV challenge replication after a single intramuscular administration

"Provided is a vaccine against respiratory syncytial virus (RSV), comprising a recombinant human adenovirus of serotype 26 or 35 that comprises nucleic acid encoding a RSV F protein or fragment thereof.

"In certain embodiments, the recombinant adenovirus comprises nucleic acid encoding RSV F protein comprising SEQ ID NO:1 of the incorporated herein Sequence Listing.

"In certain embodiments, the nucleic acid encoding RSV F protein is codon optimized for expression in human cells. In such embodiments, the nucleic acid encoding RSV F protein may comprise SEQ ID NO:2.

"In certain embodiments, the recombinant human adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and the E3 regions of the adenoviral genome.

"In certain embodiments, the recombinant adenovirus has a genome comprising, at its 5' terminal ends, the polynucleotide CTATCTAT.

"Further provided is a method for vaccinating a subject against RSV, the method comprising administering to the subject a vaccine hereof.

"In certain embodiments, the vaccine is administered intramuscularly.

"In certain embodiments, a vaccine hereof is administered to the subject more than once.

"In certain embodiments, the method for vaccinating a subject against RSV further comprises administering to the subject a vaccine comprising a recombinant human adenovirus of serotype 35 that comprises nucleic acid encoding a RSV F protein or fragment thereof.

"In certain embodiments, the method of vaccinating a subject against RSV further comprises administering RSV F protein (preferably formulated as a pharmaceutical composition, thus, a protein vaccine) to the subject.

"In certain embodiments, the method for vaccination consists of a single administration of the vaccine to the subject.

"Also provided is a method for reducing infection and/or replication of RSV in, e.g., the nasal tract and lungs of, a subject, comprising administering to the subject by intramuscular injection of a composition comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a RSV F protein or fragment thereof. This will reduce adverse effects resulting from RSV infection in a subject, and thus contribute to protection of the subject against such adverse effects upon administration of the vaccine. In certain embodiments, adverse effects of RSV infection may be essentially prevented, i.e., reduced to such low levels that they are not clinically relevant. The recombinant adenovirus may be in the form of a vaccine hereof, including the embodiments described above.

"Also provided is an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a RSV F protein or fragment thereof.

"Further provided is a method for making a vaccine against respiratory syncytial virus (RSV), comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a RSV F protein or fragment thereof, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus in a pharmaceutically acceptable composition. The recombinant human adenovirus of this aspect may also be any of the adenoviruses described in the embodiments above.

"Also provided is an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a RSV F protein or fragment thereof. The adenovirus may also be any of the adenoviruses as described in the embodiments above.

BRIEF DESCRIPTION OF THE FIGURES

"FIG. 1 shows the cellular immune response against F peptides overlapping the aa 1-252 of F and F peptides overlapping the aa 241-574 of F of mice upon immunization with different doses of rAd26 (A) and rAd35 (B) based vectors harboring the RSV F gene at 2 and 8 weeks after immunization

"FIG. 2 shows the antibody response against RSV in mice upon immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at 2 and 8 weeks after immunization.

"FIG. 3 shows the results of ratio of IgG2a vs. IgG 1 antibody response against RSV in mice upon immunization with 10.sup.10 vp of rAd26 and rAd35 based vectors harboring the RSV F gene at 8 weeks after immunization.

"FIG. 4 shows the virus neutralization capacity against RSV Long in mice upon immunization with different doses of rAd26 (A) and rAd35 (B) based vectors harboring the RSV F gene at 2 and 8 weeks after immunization.

"FIG. 5 shows the cellular immune response against (A) F peptides overlapping the aa 1-252 of F and (B) F peptides overlapping the aa 241-574 of F of mice upon prime boost immunization with rAd26 and rAd35 based vectors harboring the RSV F gene at 6 and 12 weeks after primary immunization.

"FIG. 6 shows the antibody response against RSV in mice upon prime boost immunization with rAd26 and rAd35 based vectors harboring the RSV F gene at different time points after the first immunization.

"FIG. 7 shows the virus neutralization capacity against RSV Long in mice serum upon prime boost immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at different time points after the first immunization.

"FIG. 8 shows the virus neutralization capacity against RSV B1 in mice upon prime boost immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at different time points after the first immunization.

"FIG. 9 shows the A) RSV lung titers and B) RSV nose titers in the cotton rats following prime boost immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at 5 days post challenge.

"FIG. 10 shows the induction of virus neutralizing titers following prime boost immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at A) 28 days, and B) 49 days after the first immunization.

"FIG. 11 shows the histopathological examination of the cotton rat lungs at day of sacrifice following prime boost immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene.

"FIG. 12 shows A) the RSV lung titers and B) the RSV nose titers in the cotton rats following single dose immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at 5 days post challenge, administered via different routes.

"FIG. 13 shows the induced virus neutralizing titers following single dose immunization with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at 28 and 49 days after the first immunization, administered via different routes.

"FIG. 14 shows the histopathological examination of the cotton rat lungs at day of sacrifice following single dose immunization (i.m.) with different doses of rAd26 and rAd35 based vectors harboring the RSV F gene at day of sacrifice.

"FIG. 15 shows maps of plasmids comprising the left end of the genome of Ad35 and Ad26 with the sequence encoding RSV F:

"A. pAdApt35BSU.RSV.F(A2)nat, and B. pAdApt26.RSV.F(A2)nat

"FIG. 16 shows A) the RSV lung titers and B) the RSV nose titers in the cotton rats following single dose immunization at day 0 or day 28 with different doses of rAd26 based vectors harboring the RSV F gene at 5 days post challenge. Challenge was at day 49.

"FIG. 17 shows the induction of virus neutralizing titers following single dose immunization with different doses of rAd26 harboring the RSV F gene at 49 days after immunization as described for FIG. 16.

"FIG. 18 shows the induction of virus neutralizing titers following single dose immunization with different doses of rAd26 harboring the RSV F gene during time after immunization

"FIG. 19 shows the VNA titers 49 days after against RSV Long and RSV Bwash with serum derived from cotton rats immunized with 10.sup.10 of Ad-RSV F or no transgene (Ad-e). PB: prime boost.

"FIG. 20 shows the RSV lung titers in the cotton rats following single dose immunization at day 0 with different doses of rAd26 based vectors harboring the RSV F gene at 5 days post challenge with RSV A2 or RSV B15/97.

"FIG. 21 shows the RSV nose titers in the cotton rats following single dose immunization at day 0 with different doses of rAd26 based vectors harboring the RSV F gene at 5 days post challenge with RSV A2 or RSV B15/97.

"FIG. 22 shows the VNA titers in the cotton rat serum following single dose immunization at day 0 with different doses of rAd26 based vectors harboring the RSV F gene at different time points post prime.

"FIG. 23 shows the RSV lung titers in the cotton rats following single dose immunization at day 0 with different doses of rAd26 based vectors harboring the RSV F gene at 5 days post challenge with a standard dose (10.sup.5) or a high dose (5.times.10.sup.5) RSV A2.

"FIG. 24 shows the RSV nose titers in the cotton rats following single dose immunization at day 0 with different doses of rAd26 based vectors harboring the RSV F gene at 5 days post challenge with challenge with a standard dose (10.sup.5) or a high dose (5.times.10.sup.5) RSV A2.

"FIG. 25 shows the RSV lung titers in the cotton rats following immunization at day 0 and 28 with different doses of single immunization or prime boost immunization with rAd26 based vectors harboring the RSV F gene at 5 days post challenge, with the challenge performed 210 days post immunization.

"FIG. 26 shows the VNA titers of the cotton rat serum following single dose and prime boost immunization at 140 days post immunization

"FIG. 27 shows the histopathological examination of the cotton rat lungs of sacrifice following single immunization or prime boost immunization with different doses of rAd26 based vectors harboring the RSV F gene at 2 days post challenge. Dots represent the median and whiskers the 25.sup.th and 75.sup.th percentile.

"FIG. 28 shows the histopathological examination of the cotton rat lungs of sacrifice following single immunization or prime boost immunization with different doses of rAd26 based vectors harboring the RSV F gene at 6 days post challenge. Dots represent the median and whiskers the 25.sup.th and 75.sup.th percentile.

"FIG. 29 shows the induction of virus neutralizing titers following immunization with rAd26 harboring the RSV F gene (Ad26.RSV.F) followed by boosting with Ad26.RSV.F or with adjuvanted RSV F protein (post-F).

"FIG. 30 shows the induction of IgG2a and IgG1 antibodies, and the ratio hereof, following immunization with Ad26.RSV.F followed by boosting with Ad26.RSV.F or by boosting with adjuvanted RSV F protein (post-F).

"FIG. 31 shows the production of IFN-g by splenocytes following immunization with Ad26.RSV.F followed by boosting with Ad26.RSV.F or with adjuvanted RSV F protein (post-F)."

For additional information on this patent application, see: RADOSEVIC, KATARINA; CUSTERS, JEROME H. H. V.; VELLINGA, JORT; WIDJOJOATMODJO, MYRA N. Vaccine against Rsv. Filed March 22, 2013 and posted June 5, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2690&p=54&f=G&l=50&d=PG01&S1=20140529.PD.&OS=PD/20140529&RS=PD/20140529

Keywords for this news article include: Antibodies, Biotechnology, Viruses, Genetics, Peptides, Virology, Adenovirus, Immunology, Pediatrics, Proteomics, Amino Acids, Vaccination, Gene Therapy, Immunization, Bioengineering, Blood Proteins, Viral Vaccines, Immunoglobulins, Biological Products, Crucell Holland B.v., Public Health Practice.

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Source: Gene Therapy Weekly


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