Vaccine development for pandemic influenza John Treanor University of Rochester

Antibody against the HA and NA are major components of influenza immunity Neuraminidase (NA) Hemagglutinin (HA) Cell surface receptor

Antibody against the HA and NA are major components of influenza immunity Functions of HA antibody Prevention of attachment Postattachment events Functions of NA antibody Prevention of cell spread Reduced severity of illness Other mechanisms M2 antibody Cellular responses

Pandemic influenza vaccine: Background information Pandemics result from emergence of influenza A viruses with new HA and/or NA Avian viruses are the source of H1-H16 and N1-N9 H5 and H7 viruses come in two types: highly pathogenic (HPAIV) and low pathogenicity (LPAIV) The structure of the HA cleavage site determines HP – multiple basic aa = HPAIV Antibody against the HA is neutralizing and protective – main goal of immunization is to induce HA antibody There is significant antigenic variation in the HA of current H5 viruses

Priority list of avian viruses with pandemic potential H5: HPAIV, current human cases, high pathogenicity H7: HPAIV, human outbreaks, generally mild disease H9: Some documented human cases, generally mild disease H2: No current human cases, still circulates in birds, proven pandemic potential H6: No human cases, but contributed genes to current H5 viruses

Options for Pandemic Vaccines Similarity to current vaccines Inactivated vaccine resembling currently licensed inactivated vaccine (+/- licensed adjuvant) Live vaccine resembling currently licensed live vaccine Inactivated vaccines with experimental adjuvants/route of administration Experimental approaches (DNA vaccines, peptides, vectors) CLOSER FARTHER

Strategies for production of H5 vaccine seed viruses Problem: HPAIV are lethal for eggs, must be used under high levels of containment Use antigenically related LPAIV (e.g., Duck/Singapore/97, H5N3) Use expressed recombinant protein (e.g., rHA A/HK/156/97) Use reverse genetics techniques to alter HA cleavage site (e.g., rg A/VN/1203/04 x PR8)

Antibody response to egg-grown A/Duck/Singapore/77 (H5N3) without adjuvant Study day H5N3 Neutralization GMT Nicholson et al Lancet 357:1937, 2001

Recombinant rHA H5 Vaccine H5N1 HA Gene Recombinant Baculovirus Insect cell expressing rHA RBCs Purified rHA H5 SDS-PAGE

Two doses of rH5 A/HK/156/97 vaccine resulted in a 50% MN response rate Percent responding* * 4-fold or greater increase to a titter of 1:80 with positive WB Vaccine 19:1732, 2001

sanofi pasteur H5N1 vaccine virus 1. Engineer the cleavage site A/Vietnam/1203/04 2. Change internal genes to PR8 rgA/Vietnam/1203/04xPR8

Geometric Mean Titer (GMT) Serum neutralizing antibody after one and two doses of H5N1 subvirion vaccine Geometric Mean Titer (GMT) 8 16 32 64 128 Treanor et al NEJM 354:1342; 2006

Reverse cumulative distribution of HAI antibody at day 56 (28 days after dose 2) Percent at above indicated titer 44% (34%, 55%) Reciprocal HAI titer Treanor et al NEJM 354:1342; 2006

Factors affecting response rates Receipt of TIV in the previous fall (significantly lower responses) Age > 40 years (significantly lower responses) Male (significantly lower rates) Multivariate analysis pending

Modeling of the post-vaccination MN response controlling for prior TIV, gender, dose, visit 1 result, and age p=.0003 (Adjusted for multiple comparisons) p=.0057 Adjusted ln neutralization titer at day 56 No TIV TIV DMID 04-063

Response to H5 compared to inactivated vaccine in children HAI GMT Percent responding 69% 84% 43% Percent > 40 50% 69% 44% Antigen A/Nanchang/933/95 (H3) A/Texas/36/91 (H1) A/Vietnam/1203/04 (H5) Pre 5.3 4.6 5.1 Post 28.9 70.3 27.7 Responses to H1 and H3 viruses in 36 children 6 mo to 18 mo of age after two doses of TIV (15 mcg per HA) 1996, unpublished Responses to H5 in 102 adults 18 to 64 years of age receiving two doses of 90 mcg NEJM 354: 1343, 2006

A 90 mcg dose in perspective Doses of licensed and experimental vaccines administered in multi-dose regimens to unprimed individuals Vaccine Subvirion H5N1 Tetanus Pertussis Hepatitis B HPV VLP rPA (Anthrax) rGP120 (HIV) Dose (mcg) 90 15 10-40 20-40 50-100 100-500 Comment (based on Lf) PT component of aP Experimental All vaccines, except H5N1, administered with alum or other adjuvants

Effects of dose on vaccine availability Current US manufacturing: ~60M x 45 mcg = 2700M mcg 90 mcg x 2 = 180 mcg = 15M 2-dose vaccines HHS goal requires 20-fold increase in US manufacturing capacity Persons vaccinated (M) Pandemic week

Strategies towards improved vaccination against pandemic infuenza Alternative route of administration (intradermal) Addition of adjuvants Booster doses Live vaccines

Aluminum based adjuvants Category of aluminum salts (e.g., aluminum phosphate, aluminum hydroxide) or mixtures Mechanism of action not defined, but depends on binding of antigen to the aluminum Widely used in human vaccines, including pediatrics, licensed in most countries Not currently used for inactivated influenza vaccines

Evaluation of alum adjuvanted vs unadjuvanted (aqueous) H1N1 vaccine in H1 naïve adults (1977) GMT HI Antibody (% >40) after 9 mcg split H1N1 SC (58%) (93%) (27%) (70%) 4 16 64 128 GMT Nicholson J Biolog Standard 7:123, 1979

GMT day 42 post vaccination HAI antibody Effect of aluminum hydroxide on responses to A/VN/1194/04 (H5N1) subvirion vaccine 8 16 32 64 7.5 mcg 15 mcg 30 mcg Alum HA dose GMT day 42 post vaccination HAI antibody Bresson et al Lancet 367:1367, 2006

Immunogenicity of a whole virus vaccine formulated with alum Dose of whole virion vaccine + alum Percent with > 4- fold HAI response after 2 doses Lin et al Lancet 2006

Oil-in-water emulsion – MF59 ANTIGEN No real benefits in young adults, modest improvements in flu response in elderly Increased local pain and irritation Licensed in some countries SQUALENE TWEEN 80 SPAN 65

Significant enhancement of the response to H5N3 virus with MF59 4 8 16 32 64 H5N3 Neutralization GMT Vaccine dose group Nicholson et al Lancet 357:1937, 2001

Serum HAI responses to H9N2 subvirion vaccine formulated with or without MF59 4 8 16 32 64 128 256 GMT HAI antibody day 56 Vaccine Dose Group Atmar et al CID 43:1135, 2006

Additional adjuvants in development AS03 (GSK) Adjuvant “A” (sanofi) CPG (Coley, others) ISCOM (CSL) Transdermal LT (Iomai) Others

Strategies towards improved vaccination against pandemic infuenza Alternative route of administration (intradermal) Addition of adjuvants Booster doses Live vaccines

Enhanced antibody responses following a third dose of H5N3 vaccine -/+ MF59 Microneutralization GMT Stephenson et al, Vaccine 21:1687, 2003

Evaluation of priming with an antigenic variant: schematic of study design 1998 2005 UR-98012 A/HK/156/97 rH5 DMID 05-0043 Healthy recipients of any rH5 rgA/VN/1203/04 H5-PRIMED Placebo 25 ug x 2 45 ug x 2 90 ug x 2 90 ug x 1 + 10 ug x 1 Placebo excluded 90 ug x 1 37 subjects This diagram shows a schematic of the study design. H5-primed subjects were derived from our original study, denoted as UR 98012, which was conducted in 1998. In that study, subjects were randomized to receive placebo or rH5 at either two doses of 25 mcg, two doses of 45 mcg, two doses of 90 mcg, or one dose of 90 mcg followed by one dose of 10 mcg, at intervals of 21, 28, or 42 days. Subjects who received any active vaccine were considered H5-primed and were recruited to participate in the current study (DMID 05-0043). All subjects received a single dose of 90 mcg of A/VN/1203/04 vaccine in open label fashion. Their results are compared to those of H5-naïve individuals who had received two doses of 90 mcg of A/Vietnam/1203/04 vaccine in study DMID 04-063 147 subjects DMID 04-063 Vaccine 19:1732, 2001 rgA/VN/1203/04 H5-NAIVE 90 ug x 2 103 subjects NEJM 354:1343, 2006 CLADE 3 CLADE 1

Serum neutralizing (NT) antibody responses following one or two doses of H5 vaccine in naïve subjects or following a single dose in H5 vaccine-primed subjects 4 8 16 32 64 128 256 H5 NAIVE H5 PRIMED DMID 04-063 DMID 05-0043 GMT NT Antibody 90 mcg 5.1 8.3 22.9 6.4 93.8 68.9 This slides show the neutralizing antibody responses in H5-naïve subjects shown in the left and the H5-primed subjects shown in the right, with 95% confidence intervals. Neutralizing titers after a single 90 mcg dose in H5 primed subjects were also higher than after one or even two doses of 90 mcg in vaccine naïve subjects

Advantages and disadvantages of priming Current strategies generally depend on two doses (naïve population) Logistically difficult in face of pandemic Requires twice as much vaccine Pre-priming would require less vaccine when pandemic occurs, easier response, possibly some protection Requires decision to administer potentially unnecessary vaccine Priming vaccine would not match pandemic strain, but multiple priming experiences might broaden response

Strategies towards improved vaccination against pandemic infuenza Alternative route of administration (intradermal) Addition of adjuvants Booster doses Live vaccines

Rapid attenuation of new antigenic variants by genetic reassortment PB1 PB1 PB2 PB2 PA PB1 PB2 PA NP M NS PA HA HA NA NA NP HA NA NP M M NS NS ATTENUATED DONOR VIRUS WILD-TYPE H5N1 VARIANT ATTENUATED H5N1 VACCINE VIRUS

Live vaccine is especially efficacious in unprimed, immunologically naïve subjects Attack rate (%) Antigenic relatedness of vaccine and circulating virus 89.2 (67.7 to 97.4) 79.2 (70.6 to 85.7) 16.1 (-7.7 to 34.7) Relative protective efficacy of CAIV compared to TIV A/H3N2 A/H1N1 B Belshe et al NEJM 356:685-96, 2007

Live vaccines Conventional CAIV are highly immunogenic in susceptible populations Higher levels of protection Potential use of low doses Induction of mucosal immunity might reduce shedding, halt transmission Broader cross protection Overattenuation is possible Concerns about transmission But…..

Experimental Approaches DNA Vaccines Live viral vectors (e.g., adenovirus, alphavirus replicons) Expressed protein/virus-like particles Cross protective peptides/epitopes Nasal inactivated vaccines Alternative live vaccines

H5 vaccines: summary Likely licensure of sanofi 90 mcg vaccine is only a first step Multiple simultaneous approaches to improved vaccines are being explored Advances in H5 vaccines may be translated into better control of seasonal influenza