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FluSure XP™: Research-Backed Swine Influenza Virus (SIV) Vaccine for Combating Rapidly Evolving SIV Viruses

by 5m Editor
21 November 2008, at 12:00am

Introduction of the H3N2 influenza virus into the US swine population in 1998 has resulted in the emergence of multiple reassortant swine influenza viruses (SIVs) in every geographic region of the country, writes Michael Kuhn, DVM, Pfizer Animal Health.

Since 1998, the classic H1N1 cluster has been replaced with reassortant H1N1 and H1N2 viruses with hemagglutinin (HA) and neuraminidase (NA) from classic H1N1 (cH1N1) and internal genes from the H3N2 virus (reassortant H1N1) or with HA from the cH1N1 virus and NA and internal genes from the H3N2 virus (H1N2).5,6,8-10 More recently, H1N1 and H1N2 viruses containing H1 genes closely related to human influenza virus genes (human-like H1) have entered US swine herds.9,10 Genetic changes have also occurred within the H3N2 SIV subtype, and four clusters or groups of genetically and antigenically diverse viruses have circulated in the US swine population.2,8,11,12 The Cluster I and Cluster II H3N2 viruses have been replaced, first with Cluster III viruses,12 and more recently with new viruses referred to as Cluster IV reassortants.2,9,13,14

Key Points

  • Currently, three H1 clusters and one H3 cluster of swine influenza virus (SIV) are the most prevalent SIVs co-circulating in US swine herds.1-6
  • In response to the continued antigenic drift and shift of SIV, Pfizer Animal Health scientists formulated an updated vaccine—FluSure XP—with H1 and H3 strains that demonstrated a high degree of genetic similarity and antigenic cross-reactivity with prevalent contemporary field isolates.
  • In September 2007, the USDA introduced new licensing guidelines for updating strains in currently-licensed inactivated SIV vaccines.
  • Pigs vaccinated with new FluSure XP responded with hemagglutination inhibition (HI) antibody titers to H1N1 and H3N2 vaccine viruses that were not less than titers from pigs vaccinated with legacy (original) FluSure®.7

Prevalence of various H1 and H3 viruses has been monitored by Dr. Marie Gramer and investigators at the University of Minnesota Veterinary Diagnostic Laboratory (MVDL). Genetic analyses of isolates from US swine between 2003 and 2007 indicate that currently circulating SIVs belong predominantly to three H1 clusters and one H3 cluster.1,2,9,10,14

  • Reassortant H1N1-like (rH1N1) H1N1 and H1N2 viruses
  • H1N2-like H1N1 and H1N2 viruses
  • Human-like H1N1 (hH1N1) and H1N2 viruses
  • Cluster IV (previously referred to as Cluster III variant) H3N2 viruses

Responding to the changing SIV epidemiological situation, the United States Department of Agriculture (USDA) proposed new guidelines in Veterinary Services (VS) Memorandum No. 800.111, issued in September 2007, to help accelerate the updating of strains in currently licensed inactivated SIV vaccines. By issuing these recommendations, the USDA recognized the need to react quickly to remove, replace, or add new SIV strains in response to the antigenic shift and drift occurring in the field. Under the new recommendations, manufacturers may demonstrate immunogenicity in host animals by serology rather than challenge.

Additional challenge studies and field safety studies are not required provided the production methods are not significantly altered.

This bulletin reviews the research Pfizer Animal Health conducted that led to the selection of the two H1N1 strains and one H3N2 strain used for FluSure XP, the new SIV vaccine from Pfizer Animal Health.

*Shaded cells indicate genetic cluster for the strain. Percent identity based on alignment and analysis of approximately the first 600 nucleotides of the HA gene at
MVDL.
†Selected for FluSure XP
‡Legacy FluSure H1N1; retained in FluSure XP

Selecting the FluSure XP viruses

Preparation of swine antisera from SIV viruses

The initial selection process for the new FluSure XP vaccine viruses was a collaborative effort involving the Pfizer Animal Health STOMP PLUS™ diagnostics program and the MVDL, where SIV isolates received between 2003 and 2006 were characterized by sequence analysis of the HA genes. Pfizer Animal Health scientists chose six H1N1 and eight H3N2

viruses as potential vaccine strains. These 14 isolates were used to prepare inactivated and adjuvanted monovalent vaccines, which were administered two times to 14 groups of caesarean-derived, colostrum-deprived (CDCD) 8-week-old pigs (3 to 4 pigs per group).14 Two additional groups of pigs were inoculated with inactivated and adjuvanted monovalent vaccines prepared from the legacy FluSure H1N1 and H3N2 vaccine strains; a seventeenth group was inoculated with adjuvanted negative cell culture fluids (placebo); and a final group was held as non-vaccinated controls. Pigs were vaccinated on Days 0 and 25 of the study, and serum samples collected one day prior to vaccination (Day -1) and at 1 week post-second vaccination (Day 32). Sera were sent to the MVDL to test for hemagglutination inhibition (HI) antibodies using the laboratory’s panel of SIV reference viruses.

*Shaded cells indicate genetic cluster for the strain. Percent identity based on alignment and analysis of approximately the first 600 nucleotides of the HA gene at MVDL.
†Selected for FluSure XP
‡Legacy FluSure H3N2; not retained in FluSure XP

Genetic and antigenic diversity confirmed

At the time of vaccination, the 6 H1N1 and 8 H3N2 field isolates were re-submitted to the MVDL for HA sequence analysis and subtype confirmation. Table 1 shows that isolates 030, 031, 032, 035, and 036 clustered with the newly emerging viruses most closely related to a human influenza virus (hH1N1), NY/2003; isolate 034 clustered with reassortant viruses represented by MN/2002 (rH1N1); and the legacy FluSure H1N1 (161) clustered with viruses represented by IN/2002 H1N2-like viruses. For the H3N2 viruses (Table 2), isolates 059 and 068 had the highest sequence similarity to the Cluster III viruses represented by the IL/1999 virus; isolate 038 was equally similar to the Cluster III and Cluster IV reference viruses; isolate 047 clustered with NC/1998 double reassortant (a Cluster I virus); isolates 066, 069, 070, and 071 fell with the Cluster IV (III variant) viruses represented by the MN/2004 and ONT/2005 viruses; and legacy FluSure H3N2 (158) clustered with the Cluster I viruses represented by the TX/1998 virus.14

Pre-vaccination sera from all pigs were negative for HI antibodies to all SIVs tested. On Day 32, sera from pigs in the placebo-vaccinated and non-vaccinated control groups were ≤40 to all test viruses. Titers of 20 and 40 in these control groups were to the Pfizer H3N2 and the NC/2001 viruses, which may indicate some non-specific background to these viruses.

The HI antibody responses to the H1N1 viruses were consistent with the HA genetic cluster of the virus with which they were vaccinated (Table 3). There was minimal cross-reactivity between the rH1N1 and H1N2-like H1N1 viruses and the newer hH1N1 viruses. Pigs vaccinated with the H3N2 Cluster III or Cluster IV viruses generally responded with high HI antibody titers to the Cluster III/IV viruses (Table 4). Some groups also responded with high titers to the legacy FluSure and TX/1998 Cluster I viruses. Pigs vaccinated with legacy FluSure H3N2 (158) responded with high HI titers to both Cluster I H3N2 viruses, but not to the newer and more prevalent Cluster III/IV viruses.


GMT = geometric mean titer; NTX= non treated controls
*Cluster and subtype based on PCR subtyping and sequencing of the hemagglutinin (HA) gene performed at MVDL.
†Titers less than 10 were reported as 5; .640 as 640.
‡ Selected for FluSure XP
#167 Legacy FluSure H1N1; retained in FluSure XP




GMT = geometric mean titer; NTX = nontreated controls
*Cluster and subtype based on PCR subtyping and sequencing of the hemagglutinin (HA) gene performed at MVDL.
† Titers less than 10 were reported as 5; . 640 as 640.
‡ Selected for FluSure XP
#Legacy FluSure H3N2; not retained in FluSure XP

Selecting broadly cross-protective strains

In summary, for H1 viruses, there is minimal cross-reactivity between the newer hH1N1 isolates and viruses representing the classic and older reassortant clusters, consistent with the approximate 70% to 75% sequence homology between the HA genes of these H1 clusters. Evaluation of cross-reactivity of H3N2 field isolates demonstrates antigenic differences between and within the genetic H3N2 clusters. Data from this study demonstrated a high degree of diversity within the H1N1 and H3N2 viruses currently circulating within US swine herds. The next step in the selection of vaccine strains for FluSure XP was to determine which of the H1N1 and H3N2 strains would stimulate immune responses that provide the most comprehensive cross-reactivity against the predominant SIVs currently co-circulating in US swine. Sera collected at 13 days following second vaccination (Day 38) from the same groups of vaccinated CDCD pigs were pooled by treatment group and tested against a full panel of H1 or H3 viruses.14

Table 5 presents results of the HI cross-reactivity of the serum pools to homologous H1N1 viruses. Pigs vaccinated with hH1N1 viruses (030, 031, 032, 035, 036) responded with antibodies that cross-reacted with high titers (≥160) to all of the hH1N1 viruses; pigs vaccinated with the rH1N1 virus (034) had a high titer (640) to the homologous virus and a lower titer to the H1N2-like virus; and pigs vaccinated with the legacy FluSure H1N1 virus (161) had a high titer only to the homologous virus

Neg = negative control pigs vaccinated with placebo or non-vaccinated (NTX) pigs Bold = . 4-fold increase over pre-vaccination titers, generally considered as a positive HI titer in field sera; shaded cell = homologous titer.
Replicates were run on each pool and the mean titer reported.
*Genetic cluster and subtyping based on hemagglutination (HA) gene sequence and PCR subtyping from MVDL that was completed on cloned viruses used in this study.
†Selected for FluSure XP
‡Legacy FluSure H1N1; retained in FluSure XP


Neg = negative control pigs vaccinated with placebo or non-vaccinated (NTX) pigs.
Bold = . 4-fold increase over pre-vaccination titers, generally considered as a positive HI titer in field sera; shaded cell = homologous titer.
Replicates were run on each pool and the mean titer reported.
*Genetic cluster and subtyping based on hemagglutination (HA) gene sequence and PCR subtyping from MVDL that was completed on cloned viruses used in this
study.
†Selected for FluSure XP
‡Legacy FluSure H3N2; not retained in FluSure XP

Table 6 presents results of the HI cross-reactivity of the treatment serum pools to homologous H3N2 viruses. Cross-reactivity between the newer Cluster III and Cluster IV H3N2 viruses generally followed their genetic groupings but some differences in responses were evident. Groups vaccinated with some viruses (047, 066, 069, and 071) had HI antibodies that reacted with all Cluster III or Cluster IV viruses tested, while others (038, 059, 068, and 070) had antibodies that did not cross-react with some of the test viruses. The legacy FluSure H3N2 vaccine group (158) responded with a high titer (960) only to the homologous virus.

Based on the most recent prevalence data, HA gene sequence homology data, and hemagglutination inhibition antibody titer studies described above, the strains selected for Pfizer Animal Health’s updated trivalent FluSure XP vaccine were:

  • the legacy FluSure H1N1 isolate 161
  • the human-like H1N1 isolate 031, and
  • the Cluster IV H3N2 isolate 069.

Studies conducted at the MVDL and Pfizer Animal Health showed that these three viruses are genetically similar to, and broadly cross-reactive with, a panel of contemporary field isolates representing the major genetic clusters now co-circulating in the North American swine herd population. Additional factors evaluated in selecting the three viruses for the new trivalent FluSure XP formulation were current SIV epidemiological data and the results of previous efficacy studies conducted with the legacy FluSure product.1,2,9,10,14,15

Establishing the efficacy of FluSure XP

The pivotal immunogenicity study described below was conducted to establish vaccine efficacy and to assess whether new FluSure XP, with its newly updated SIV strains, generated immune responses that were not less than those produced by the legacy FluSure formulation.7 Results of the study summarized below in Table 7 demonstrated that pigs vaccinated with the new trivalent FluSure XP responded with HI antibody titers to the vaccine viruses that were similar to the HI antibody titers of pigs vaccinated with the legacy bivalent FluSure, thereby fulfilling the recommendations of VS Memorandum No. 800.111. In all pigs in all treatment groups, individual HI titers to the vaccine viruses were ≥ 40, the titer considered to be protective against swine influenza.16,17

Summary of the Study Evaluating the Immunogenicity of New Trivalent FluSure XP7

Study Design

PIGS: 65 clinically healthy, 3-week-old cross-bred pigs with no history of exposure to or vaccination against SIV were allocated to one of four treatment groups and a NTX control group (15 pigs/group; 5 NTX controls).

  • T1 Legacy FluSure (minimum immunizing dose)
  • T2 FluSure XP (minimum immunizing dose)
  • T3 FluSure XP (medium dose)
  • T4 FluSure XP (high dose)

VACCINATION PROTOCOL: Pigs in T1-T4 were vaccinated intramuscularly on Day 0 and Day 14 with a 2 mL dose rehydrated with Amphigen® adjuvant.

ASSESSMENTS: HI antibody determinations were made on Day -1, 13, 28 serum samples.

CRITERIA: To be considered serologically similar, FluSure XP had to demonstrate similar geometric mean titers (GMTs) when compared to GMTs obtained with the legacy FluSure product for each of the H1N1 and H3N2 fractions contained in the vaccine.

Study Results

  • Only the Day 28 data for the legacy FluSure minimum immunizing dose group (T1) and the group vaccinated with the minimum dose of FluSure XP (T2) are shown.
  • All pigs had HI titers less than 10 at the time of first vaccination.
  • The NTX control pigs showed no evidence of SIV exposure at the Day 13 and Day 28 blood collections.
  • H1N1 antibody responses to legacy FluSure and FluSure XP were similar.
  • For the legacy FluSure H1N1 virus, the GMT of the FluSure XP group , containing both the current and new viruses, all fell within the 95% Confidence Interval (CI) of the GMT of the legacy FluSure group (see description of 95% CI below).
  • HI titers to the legacy FluSure H1N1 virus of indi vidual pigs were ≥ 40; this titer has been shown to be protective against swine influenza.16,17
  • All FluSure XP vaccinated pigs had HI titers . 40 to the new hH1N1 virus.
  • H3N2 antibody responses to legacy FluSure and FluSure XP were similar.
  • The GMT of the FluSure XP group to the homologous H3N2 vaccine strain all fell within the 95% CI of the GMT of pigs vaccinated with the legacy FluSure product.
  • The HI titers of individual pigs were . 40 to the H3N2 virus contained in the vaccine.
  • The GMTs of all swine influenza viral fractions were not less than those previously demonstrated to be efficacious for the legacy FluSure product.15

GMT = geometric mean titer
*T1 and T2 targeted at minimum immunizing dose levels.


Description of 95% Confidence Interval:

One measure of similarity is the evaluation of the confidence interval (CI). A confidence interval for a mean specifies a range of values within which the unknown population parameter, in this case the mean, may lie. We calculate these intervals for different confidence levels, depending on how precise we want to be. The 95% CI is a measure that we are 95 percent sure that the true mean of the HI antibody titers on Day 28 lies between two values.

Updating protection against swine influenza

New Trivalent FluSure XP

In response to the changing SIV epidemiological situation, the USDA introduced new guidelines to facilitate updating of strains in currently-licensed inactivated SIV vaccines. FluSure XP is the first SIV vaccine to be licensed under these guidelines. The hH1N1, H1N2-like, and Cluster IV H3N2 vaccine strains selected for FluSure XP were chosen because they were genetically similar to and broadly crossreactive with a panel of contemporary field isolates representing the major genetic clusters that are currently circulating in North American swine herds. On the basis of epidemiological data, HI cross-reactivity, and previous efficacy studies conducted with legacy FluSure, two H1N1 viruses (FluSure H1N1 isolate 161 and human-like H1N1 isolate 031) and one H3N2 virus (Cluster IV H3N2 isolate 069) were selected for the new FluSure XP vaccine formulation. The legacy FluSure H1N1 virus belongs to the genetic group of H1N2-like swine influenza viruses that first appeared in US swine herds in 1999/2000.5,8,18,19 The human-like H1N1 selected for FluSure XP has more than 95% HA sequence similarity with the MVDL human H1N1 reference virus (NY/2003) and stimulated antibody titers ranging from 640 to 1280 against a panel of currently circulating hH1N1 viruses. The legacy FluSure Cluster I H3N2 virus has been replaced in FluSure XP with an H3N2 Cluster IV isolate that has 95% to 99% HA sequence similarity to Cluster III and Cluster IV H3N2 reference viruses at the MVDL and stimulates high HI cross-reactivity with currently circulating Cluster IV field isolates.

Updating protection against swine influenza

In the pivotal immunogenicity study conducted for licensing, pigs vaccinated with trivalent FluSure XP responded with antibody titers to the H1N1 vaccine viruses that were not less than titers from pigs vaccinated with bivalent legacy FluSure.7,15 Pigs vaccinated with FluSure XP also had titers (. 40) to the H1N1 and H3N2 vaccine viruses that have been reported to protect swine against influenza.16,17 Previous vaccination and challenge studies conducted with the legacy FluSure vaccine established that the H1N2-like vaccine virus helps protect pigs against disease caused by H1N2-like, rH1N1, and cH1N1 SIVs.1,15,18-20 Thus, current and previous data demonstrate the efficacy of FluSure XP against viruses representing the predominant H1 and H3 subtypes now co-circulating in the US swine population.

Producers and veterinarians often experience great frustration in attempting to tailor SIV vaccination protocols to the complex mix of SIVs co-circulating and prevalent in swine herds at any given time. Due to this situation, SIV vaccine manufacturers increasingly will need to rely upon diligent disease monitoring, coupled with serological and viral surveillance, to ensure that they update vaccines to meet the antigenic drift and shift that results in evolutional changes to the viruses. Under the new USDA guidelines, manufacturers now have the opportunity to address vaccine updates in a more timely and agile manner.

Committed to early detection of where, when, and which SIVs are circulating in US swine herds, Pfizer Animal Health has assembled the SIV Surveillance Team to assist producers and veterinarians with their ongoing efforts to control swine influenza. This cadre of dedicated scientists has been formed not only to monitor changes in the prevalence of current and emerging SIV strains but also to assess market needs and to assist in updating future formulations of FluSure XP. Their efforts will facilitate prompt detection and characterization of SIVs and ultimately will accelerate implementation of effective control programs based on the use of vaccines with the closest possible match between vaccinal strains and circulating disease strains.

In addition to the formation of the SIV Surveillance Team and release of the new, trivalent FluSure XP, Pfizer Animal Health has created the XP Defense System, designed to assist producers and veterinarians and provide solutions to their SIV-specific problems. For additional information on Pfizer’s XP Defense System and for help in determining how this system might be used to address the specific needs of your operation, contact your Pfizer Swine Technical Services veterinarian or sales representative and visit FluSureXP.com.

References

1. Gramer M, Rossow K. Epidemiology of swine influenza and implications of reassortment. Allen D. Leman Conference 2004;69-73.

2. Gramer M, Nitzel G, Wicklund E, et al. Serologic cross-reactivity between reference strains and field isolates representing different genetic clusters of H1N1 and H3N2 swine influenza virus. American Association of Swine Veterinarians 2008;99-101.

3. Gramer MR, Lee JH, Choi YK, et al. Serologic and genetic characterization of North American H3N2 swine influenza A viruses. Can J Vet Res 2007;71:201-206.

4. Vincent AL, Lager KM, Ma W, et al. Evaluation of hemagglutinin subtype 1 swine influenza viruses from the United States. Vet Microbiol 2006;118:212-222.

5. Karasin AI, Anderson GA, Olsen CW. Genetic characterization of an H1N2 influenza virus isolated from a pig in Indiana. J Clin Microbiol 2000;38:2453-2456.

6. Karasin AI, Carman S, Olsen CW. Identification of human H1N2 and human-swine reassortant H1N2 and H1N1 influenza A viruses among pigs in Ontario, Canada (2003 to 2005). J Clin Microbiol 2006;44:1123-1126.

7. Data on file, Study Report No. 3125R-60-07-503, Pfizer Inc.

8. Webby RJ, Rossow K, Erickson G. Multiple lineages of antigenically and genetically diverse influenza A virus co-circulate in the United States swine population. Virus Res 2004;103:67-73.

9. Gramer M. SIV: An update on circulating strains, advances in diagnostic tests and interpretation of test results. In “Backseat” Respiratory Disease. Pre-conference seminar at AASV, Orlando, Florida. 2007;38:11-17.

10. Gramer M. Reassortant human/swine H1N1 and H1N2 influenza virus infections in US swine. American Association of Swine Veterinarians 2006;463-464.

11. Webby RJ, Swenson SL, Krauss SL, et al. Evolution of swine H3N2 influenza viruses in the United States. J Virol 2000;74(18):8243-8251. 12. Richt JA, Lager KM, Janke BH, et al.

Pathogenic and antigenic properties of phylogenetically distinct reassortant H3N2 swine influenza viruses cocirculating in the United States. J Clin Microbiol 2003;41(7):3198-3205.

13. Olsen CW, Karasin AI, Carman S, et al. Triple reassortant H3N2 influenza A viruses, Canada, 2005. Emerg Infect Dis 2006;12(7):1132-1135.

14. Data on file, Study Report No. 3121R-60-06-451, Pfizer Inc.

15. Data on file, Study Report No. 2124H-60-01-060, and No. 2124H-60-01-009, Pfizer Inc.

16. Wasmoen T. Immune response to swine influenza vaccination. In: Tracking and Managing SIV. Pre-conference seminar at Allen D. Leman Conference. Minneapolis, Minnesota. 2001;23-31.

17. Kuhn M, Ficken MD. Protection against H1N1 and H3N2 swine influenza virus challenges by maternal antibody. American Association of Swine Veterinarians 2003;231-233.

18. Kitikoon P, Thacker E, Nilubol D, et al. Immune response and effect of maternal antibody interference on vaccination with a bivalent swine influenza virus vaccine. American Association of Swine Veterinarians 2005;363-365.

19. Rapp-Gabrielson VJ, Wicklund E, Ficken M. Efficacy of FluSure® against challenge with heterologous reassortant swine influenza H1N1 viruses. Allen D. Leman Conference 2005;32 (Supplement):14.

20. Data on file, Study Report No. 2124H-60-01-061, Pfizer Inc.

October 2008