Efficacy of live attenuated vaccines against 2009 pandemic H1N1 influenza in ferrets

The advent of the H1N1 influenza pandemic (pH1N1) in 2009 triggered the rapid production of pandemic influenza vaccines, since seasonal influenza vaccines were expected and demonstrated not to provide significant cross-protection against the newly emerged pandemic virus. To increase vaccine production capacity and further evaluate the effectiveness of different candidate pandemic influenza vaccines, the World Health Organization stimulated the evaluation of different vaccination concepts including the use of live attenuated influenza vaccines (LAIVs). Therefore, we have immunized ferrets intranasally with a single dose of pH1N1-LAIV from different manufacturers. They all induced adequate serum HI antibody titers in the ferrets and protected them against intratracheal wild-type pH1N1 virus challenge: pH1N1 virus replication in the upper respiratory tract and lungs was reduced and no disease signs or severe broncho-interstitial pneumonia were observed in any of the vaccinated ferrets. These data together with the relatively efficient production process emphasize the potential of the LAIV concept for pandemic preparedness.     

Cold-adapted X-31 live attenuated 2009 pandemic H1N1 influenza vaccine elicits protective immune responses in mice and ferrets

The 2009 pandemic influenza H1N1 (pdmH1N1) is characterized by rapid transmission among humans and disproportionate infection to children and young adults. Although the pdmH1N1 demonstrated less lethality than initially expected and has now moved into its post-pandemic period, it remains highly possible that through antigenic shift or antigenic drift the pdmH1N1 might re-emerge in the future as a more virulent strain than before, underscoring the need for vaccination prior to an outbreak. Using X-31 ca as a backbone strain, we generated a live attenuated pdmH1N1 vaccine and evaluated its potential as a safe and effective vaccine using mouse and ferret models. Despite an acceptable level of attenuation phenotypes, single dose of immunization with the vaccine efficiently stimulated both systemic and mucosal antibody responses and provided complete protection against lethal challenge with wild type pdmH1N1 virus, even at the lowest immunization dose of 10³ PFU. The promising results of safety, immunogenicity, and protective efficacy of the vaccine not only contribute to expanding the repertoire of live vaccines as a judicious choice for pandemic H1N1 preparedness, but also suggest the great potential of X-31 ca donor strain to serve as reliable platform for generating diverse live vaccine constructs against seasonal influenza viruses and other pandemic strains.     

NS-based live attenuated H1N1 pandemic vaccines protect mice and ferrets

Although vaccines against influenza A virus are the most effective method to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. We generated live attenuated human influenza A vaccines (LAIVs) by rationally engineering mutations directly into the genome of a pandemic-H1N1 virus. Two LAIVs (NS1-73 and NS1-126) were based on the success of LAIVs for animal influenza A viruses. A third candidate (NSΔ5) is a unique NS-mutant that has never been used as a LAIV. The vaccine potential of each LAIV was determined through analysis of attenuation, interferon production, immunogenicity, and their ability to protect mice and ferrets. This study demonstrates that NSΔ5 is an ideal LAIV candidate, provides important information on the effects that different NS mutations have on the pandemic-H1N1 virus and shows that LAIVs can be engineered directly from the genomes of emerging/circulating influenza A viruses.     

An M2 cytoplasmic tail mutant as a live attenuated influenza vaccine against pandemic (H1N1) 2009 influenza virus

The 2009 influenza pandemic brought home the importance of vaccines in infection control. Previously, we demonstrated an M2 cytoplasmic tail mutant H5N1 influenza virus could serve as a live-attenuated vaccine. Here, we adapted that strategy, generating a mutant pandemic (H1N1) 2009 virus that grew well in cell culture, but replicated less well in mice than did wild-type virus. The mutant virus elicited sterile immunity in mice, completely protecting them from challenge with a pandemic (H1N1) 2009 virus. Our results indicate that M2 cytoplasmic tail mutants are suitable for live-attenuated vaccines against pandemic viruses.     

Efficacy of live attenuated influenza vaccine against influenza illness in children as a function of illness severity

A recent study of inactivated influenza vaccine (IIV) in children aged 3–8 years demonstrated higher efficacy against moderate/severe influenza. A meta-analysis of all previous published randomized clinical trials of live attenuated influenza vaccine (LAIV) that collected information on illness severity in children aged 24–71 months was conducted. Moderate/severe influenza was defined as fever >39 °C, acute otitis media, or lower respiratory tract illness; other cases were classified as milder influenza. LAIV efficacy versus placebo was 95.4% [95% confidence interval: 88.5, 98.1] (year 1) and 88.5% [77.4, 94.9] (year 2) against moderate/severe influenza and 91.4% [77.9, 96.7] (year 1) and 84.2% [56.7, 94.3] (year 2) against milder influenza. The relative efficacy of LAIV versus IIV was 52.2% [31.6, 66.6] for moderate/severe influenza and 45.0% [28.6, 57.5] for milder influenza. Efficacy against all influenza illnesses, regardless of severity, is critical to prevent influenza illness and transmission in the community.     

Efficacy of live attenuated influenza vaccine in children against influenza B viruses by lineage and antigenic similarity

Seasonal influenza vaccines, including live attenuated influenza vaccine (LAIV), contain three vaccine strains (two type A and one type B). Ideally, the hemagglutinin antigens of the recommended vaccine strains are antigenically similar to epidemic wild-type strains; in actuality, the antigenic match between circulating and vaccine strains each year can vary significantly owing to intermittent genetic reassortment and continuous antigenic drift. For influenza B, antigenic relatedness is further complicated by the existence of two distinct lineages. Consequently, the influenza B vaccine component can be of a completely different antigenic lineage from the circulating epidemic strains. Using data from nine randomized clinical trials in young children (6 months to 6 years of age), vaccine efficacy of LAIV against influenza B strains was assessed across this spectrum of antigenic relatedness. In an integrated analysis, vaccine efficacy of two doses of LAIV in vaccine-naive children was 86% against B strains of the same lineage and closely matched to the vaccine strain, 55% against strains of the same lineage but antigenically drifted from the vaccine strain, and 31% against strains of the opposite B lineage and antigenically unrelated to the vaccine strain. These data provide a more accurate assessment of the protection provided by the current trivalent vaccine and highlight the need for vaccination strategies that provide enhanced protection against both lineages of influenza B such as a quadrivalent influenza vaccine.     

Genetic stability of live attenuated vaccines against potentially pandemic influenza viruses

BackgroundEnsuring genetic stability is a prerequisite for live attenuated influenza vaccine (LAIV). This study describes the results of virus shedding and clinical isolates’ testing of Phase I clinical trials of Russian LAIVs against potentially pandemic influenza viruses in healthy adults.MethodsThree live attenuated vaccines against potentially pandemic influenza viruses, H2N2 LAIV, H5N2 LAIV and H7N3 LAIV, generated by classical reassortment in eggs, were studied. For each vaccine tested, subjects were randomly distributed into two groups to receive two doses of either LAIV or placebo at a 3:1 vaccine/placebo ratio. Nasal swabs were examined for vaccine virus shedding by culturing in eggs and by PCR. Vaccine isolates were tested for temperature sensitivity and cold-adaptation (ts/ca phenotypes) and for nucleotide sequence.ResultsThe majority of nasal wash positive specimens were detected on the first day following vaccination. PCR method demonstrated higher sensitivity than routine virus isolation in eggs. None of the placebo recipients had detectable vaccine virus replication.All viruses isolated from the immunized subjects retained the ts/ca phenotypic characteristics of the master donor virus (MDV) and were shown to preserve all attenuating mutations described for the MDV. These data suggest high level of vaccine virus genetic stability after replication in humans.During manufacture process, no additional mutations occurred in the genome of H2N2 LAIV. In contrast, one amino acid change in the HA of H7N3 LAIV and two additional mutations in the HA of H5N2 LAIV manufactured vaccine lot were detected, however, they did not affect their ts/ca phenotypes.ConclusionsOur clinical trials revealed phenotypic and genetic stability of the LAIV viruses recovered from the immunized volunteers. In addition, no vaccine virus was detected in the placebo groups indicating the lack of person-to-person transmission.LAIV TRIAL REGISTRATION at ClinicalTrials.gov: H7N3-NCT01511419; H5N2-NCT01719783; H2N2-NCT01982331.     

流感减毒活疫苗预防儿童季节性流感保护效果的Meta分析

目的评价流感减毒活疫苗(LAIV)预防2~17岁儿童季节性流感的保护效果。方法通过Web of Science、PubMed和ScienceDirect数据库,检索2003年1月至2018年11月期间发表的、研究设计为病例对照的、关于研究LAIV预防儿童季节性流感保护效果的相关文献,采用Stata 13.1软件对纳入文献进行Meta分析。结果共纳入文献14篇,均为检测阴性设计(Test-negative design,TND)研究。结果显示LAIV预防儿童季节性流感的保护效果为49%(95%CI:40%~57%)。亚组分析发现:LAIV预防A(H1N1)pdm09型、A(H3N2)型和B型流感的保护效果分别为35%(95%CI:5%~56%)、35%(95%CI:21%~46%)和71%(95%CI:55%~82%);三价LAIV和四价LAIV在儿童中的保护效果分别为56%(95%CI:48%~63%)和44%(95%CI:27%~57%);LAIV在欧洲地区和北美洲地区的保护效果分别为65%(95%CI:47%~77%)和46%(95%CI:36%~55%)。结论LAIV对2~17岁儿童季节性流感具有一定的预防作用。     

H7N3 live attenuated influenza vaccine has a potential to protect against new H7N9 avian influenza virus

After recent emergence of new avian influenza A(H7N9) viruses in humans many people and Governments are asking about H7 influenza vaccine which could provide cross-protection against new viruses, until H7N9 vaccine is prepared from a relevant strain. Here we scientifically justify that available H7N3 live attenuated influenza vaccine (LAIV) can be protective against H7N9 viruses due to the presence of conserved immune epitopes in its hemagglutinin. We used Immune Epitope Database analysis resource to predict B-cell and CTL epitopes distributed across H7N3 HA molecule and assessed their identity with new H7N9 viruses at near 70% and 60% of the epitopes, respectively. In addition, we tested serum samples of volunteers participated in phase I clinical trial of H7N3 LAIV for the presence of anti-H7N9 hemagglutination-inhibition and neutralizing antibodies and found seroconversions in 44.8% of vaccinated persons, which suggests the potential of H7N3 LAIV to protect against new H7N9 avian influenza viruses.     

Restricted replication of the live attenuated influenza A virus vaccine during infection of primary differentiated human nasal epithelial cells

Live Attenuated Influenza Vaccine (LAIV) strains are associated with cold adapted, temperature sensitive and attenuated phenotypes that have been studied in non-human or immortalized cell cultures as well as in animal models. Using a primary, differentiated human nasal epithelial cell (hNEC) culture system we compared the replication kinetics, levels of cell-associated viral proteins and virus particle release during infection with LAIV or the corresponding wild type (WT) influenza viruses. At both 33 °C and 37 °C, seasonal influenza virus and an antigenically matched LAIV replicated to similar titers in MDCK cells but seasonal influenza virus replicated to higher titers than LAIV in hNEC cultures, suggesting a greater restriction of LAIV replication in hNEC cultures. Despite the disparity in infectious virus production, the supernatants from H1N1 and LAIV infected hNEC cultures had equivalent amounts of viral proteins and hemagglutination titers, suggesting the formation of non-infectious virus particles by LAIV in hNEC cultures.     

Recombinant H1N1 virus-like particle vaccine elicits protective immunity in ferrets against the 2009 pandemic H1N1 influenza virus

The pandemic virus of 2009 (2009 H1N1) continues to cause illness worldwide, especially in younger age groups. The widespread H1N1 virus infection further emphasizes the need for vaccine strategies that are effective against emerging pandemic viruses and are not dependent on the limitations of traditional egg-based technology. This report describes a recombinant influenza virus-like particle (VLP) vaccine consisting of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins of influenza A/California/04/2009 (H1N1) virus. Influenza H1N1 VLPs with a diameter of approximately 120 nm were released into the culture medium from Sf9 insect cells infected with recombinant baculovirus coexpressing HA, NA, and M1 proteins. Purified recombinant H1N1 VLPs morphologically resembled influenza virions and exhibited biological characteristics of influenza virus, including HA and NA activities. In the ferret challenge model, 2009 influenza H1N1 VLPs elicited high-titer serum hemagglutination inhibition (HI) antibodies specific for the 2009 H1N1 virus and inhibited replication of the influenza virus in the upper and lower respiratory tract tissues following A/Mexico/4482/09 (H1N1) virus challenge. Moreover, a single 15 μg dose of H1N1 VLPs resulted in complete virus clearance in the ferret lung. These results provide support for the use of recombinant influenza VLP vaccine as an effective strategy against pandemic H1N1 virus.     

Immunogenicity and protective efficacy of an elastase-dependent live attenuated swine influenza virus vaccine administered intranasally in pigs

Influenza A virus is an important respiratory pathogen of swine that causes significant morbidity and economic impact on the swine industry. Vaccination is the first choice for prevention and control of influenza infections. Live attenuated influenza vaccines (LAIV) are approved for use in humans and horses and their application provides broad protective immunity, however no LAIV against swine influenza virus (SIV) exists in the market. Previously we reported that an elastase-dependant mutant SIV A/Sw/Sk-R345V (R345V) derived from A/Sw/Saskatchewan/18789/02 (H1N1) (SIV/Sk02) is highly attenuated in pigs. Two intratracheal administrations of R345V induced strong cell-mediated and humoral immune responses and provided a high degree of protection to antigenically different SIV infection in pigs. Here we evaluated the immunogenicity and the protective efficacy of R345V against SIV infection by intranasal administration, the more practical route for vaccination of pigs in the field. Our data showed that intranasally administered R345V live vaccine is capable of inducing strong antigen-specific IFN-γ response from local tracheo-bronchial lymphocytes and antibody responses in serum and respiratory mucosa after two applications. Intranasal vaccination of R345V provided pigs with complete protection not only from parental wild type virus infection, but also from homologous antigenic variant A/Sw/Indiana/1726/88 (H1N1) infection. Moreover, intranasal administration of R345V conferred partial protection from heterologous subtypic H3N2 SIV infection in pigs. Thus, R345V elastase-dependent mutant SIV can serve as a live vaccine against antigenically different swine influenza viruses in pigs.     

Genetic sequence analysis of influenza viruses and illness severity in ill children previously vaccinated with live attenuated or inactivated influenza vaccine

In a large comparative study in 2004–2005, children aged 6–59 months vaccinated with live attenuated influenza vaccine (LAIV) experienced 55% fewer cases of culture-confirmed influenza illness compared with trivalent inactivated influenza vaccine (TIV) recipients. To better understand the characteristics of the breakthrough influenza illnesses, we analyzed the HA1 genetic sequence for all available samples and examined disease severity by strain and treatment group. All 48 A/H1N1 viruses were well-matched to the vaccine, whereas all 276 A/H3N2 viruses and 349 (96%) influenza B viruses were mismatched to the vaccine. The incidence of fever or lower respiratory illness did not differ by strain; however, LAIV recipients had less febrile disease and fewer lower respiratory illnesses than TIV recipients. Viruses of each influenza B lineage caused more illnesses than A/H1N1 viruses; strategies to enhance protection against multiple influenza B lineages should be pursued.     

Efficacy of live attenuated influenza vaccine in children: A meta-analysis of nine randomized clinical trials

Nine randomized clinical trials, including approximately 25,000 children aged 6–71 months and 2000 children aged 6–17 years, have evaluated the efficacy of live attenuated influenza vaccine (LAIV) against culture-confirmed influenza as compared to placebo or trivalent inactivated vaccine (TIV). We conducted meta-analyses, based on Mantel–Haenszel relative risks from fixed effect models, to provide an estimate of vaccine efficacy (VE). Relative to placebo, year 1 VE for two doses in vaccine-naïve young children was 77% (95% CI: 72%, 80%; P < 0.001) against antigenically similar strains and 72% against strains regardless of antigenic similarity. Efficacy was 85%, 76%, and 73% against antigenically similar A/H1N1, A/H3N2, and B, respectively. Year 1 VE of one dose against antigenically similar strains in vaccine-naive children was 60%; efficacy of one dose in previously vaccinated children in year 2 of the various studies was 87%. In head-to-head trials comparing two doses of TIV and LAIV, vaccine-naïve children who received two doses of LAIV experienced 46% fewer cases of influenza illness caused by antigenically similar strains. Similarly, for studies including older children who had been previously vaccinated, those receiving one LAIV dose experienced 35% fewer cases of influenza illness than those receiving one TIV dose. LAIV showed high VE versus placebo with no evidence of difference by age or by circulating subtype. In these studies, LAIV was more effective than TIV.     

Efficacy of the NS1-truncated live attenuated influenza virus vaccine for swine against infection with viruses of major North American and European H3N2 lineages

Control of swine influenza A virus (swIAV) in North America and Europe is complicated because multiple antigenically distinct swIAV strains co-circulate in the field, and no vaccine is available that can provide broad cross-protection against all these swIAVs. In 2017, the first live attenuated influenza vaccine (LAIV) for swine was licensed in the US. The non-structural protein 1 (NS1)-truncated cluster I H3N2 strain A/swine/Texas/4199-2/98 NS1del126 (TX98 LAIV) in this vaccine provides partial cross-protection against heterologous North American cluster II and IV H3N2 swIAV strains. Its efficacy against European or more recent North American H3N2 lineages remains to be investigated. In this study, we evaluated the level of cross-protection against heterologous IAVs representative of the major H3N2 swIAV lineages in Europe and North America. TX98 LAIV prevented both nasal shedding and replication in the lungs of a North American cluster IV H3N2 swIAV for 2/4 pigs, prevented considerable nasal shedding of a North American novel human-like H3N2 swIAV for 2/4 pigs, and reduced replication of a European H3N2 swIAV in the lower respiratory tract to minimal titers for 1/3 pigs. Although TX98 LAIV elicited neutralizing antibodies against the homologous virus in serum and to a lesser extent in nose and lungs, no significant cross-reactive antibody titers against the heterologous swIAVs were detected. Partial cross-protection therefore likely relies on cellular and mucosal immune responses against conserved parts of the swIAV proteins. Since TX98 LAIV can offer partial protection against a broad range of H3N2 swIAVs, it might be a suitable priming vaccine for use in a heterologous prime-boost vaccination strategy.     

Intranasal seasonal influenza vaccine and a TLR-3 agonist,rintatolimod, induced cross-reactive IgA antibody formation against avian H5N1 and H7N9 influenza HA in humans

The intranasal use of rintatolimod, a specific TLR-3 agonist, combined with trivalent seasonal influenza vaccine generated cross-protection against highly pathogenic H5N1 avian influenza in mice. The purpose of this clinical trial is to assess the safety and impact of rintatolimod on intranasal influenza vaccine in healthy adults. During Stage I of this Phase I/II clinical trial, 12 volunteers were immunized intranasally with 3 doses of FluMist^® seasonal influenza vaccine on Days 0, 28, and 56 followed by intranasal rintatolimod (50 μg, 200 μg, or 500 μg) 3 days later. Parotid saliva and nasal wash samples were collected at baseline and on Days 25, 53, 84, and 417. The samples were tested for IgA and IgG specific antibodies (Ab) directed against the homologous FluMist^® viral hemagglutinins (HAs). In addition, viral specific responses against influenza A HAs were tested for IgA Ab cross-reactivity against 3 H5 clades: HA (H5N1) A/Indonesia/5/2005, HA (H5N1) A/Hong Kong/483/97 and HA (H5N1) A/Vietnam/1194/2004, as well as, two H7 strains, HA (H7N9) A/Shanghai/2/2013 and HA (H7N3) A/chicken/Jalisco/CPA1. The combination of the intranasal FluMist^® along with the rintatolimod generated specific secretory IgA responses of at least 4-fold over baseline against at least one of the homologous vaccine strains included in the vaccine in 92% of the vaccinees. Additionally, this vaccination strategy induced cross-reactive secretory IgA against highly pathogenic avian influenza virus strains H5N1, H7N9, and H7N3 with pandemic potential for humans. The combination of rintatolimod and FluMist^® was well-tolerated.     

Evaluation of a candidate live attenuated influenza vaccine prepared in Changchun BCHT (China) for safety and efficacy in ferrets

We evaluated the safety and efficacy of a live attenuated influenza vaccine (LAIV) product in ferrets. The BCHT LAIV product was significantly less virulent than wild-type H1N1 virus, when evaluated by comparing virus shedding and histopathologic lesions. The data indicated strong evidence for an attenuated phenotype of LAIV. Furthermore, the vaccine induced robust humoral immune responses in seronegative ferrets, and protected ferrets against development of fever, weight loss and turbinate inflammatory lesions after challenging with H3N2 wide-type influenza virus. Thus, the BCHT LAIV product was safe in healthy seronegative ferrets and protected ferrets against infection of H3N2 influenza virus.     

The role of nasal IgA in children vaccinated with live attenuated influenza vaccine

Background

Immunoglobulin A (IgA) is the predominant antibody produced in response to mucosal infections. The role of IgA in providing protection against influenza in children vaccinated with live attenuated influenza vaccine (LAIV) has not been well described.

Methods

Nasal IgA responses were assessed using data from 3 prospective, 2-year, randomized studies comparing LAIV with placebo in children 6–36 months of age. In each study, samples were collected in a subset of patients; a new cohort was enrolled each year. Ratios of strain-specific nasal IgA to total nasal IgA were calculated and prevaccination to postvaccination geometric mean fold-rises (GMFRs) were evaluated. Mean postvaccination IgA ratios were compared for subjects with and without confirmed influenza illness by study and in pooled analyses.

Results

Across studies, a higher percentage of children receiving LAIV had a ≥2-fold increase in strain-specific IgA ratio compared with placebo recipients. GMFRs after LAIV in years 1 and 2 ranged from 1.2 to 6.2, compared with 0.5–2.2 among placebo recipients. Similar responses were observed in subjects who were baseline seronegative and seropositive based on serum hemagglutination inhibition antibody titers. In years 1 and 2, the mean postvaccination strain-specific to total IgA ratio was 3.1-fold (P < 0.01) and 2.0-fold (P < 0.03) higher among LAIV recipients with no evidence of culture-confirmed influenza illness compared with LAIV recipients who developed culture-confirmed influenza illness; a similar and consistent trend was observed for each individual study and type/subtype.

Conclusions

The current analysis demonstrates that nasal IgA contributes to the efficacy of LAIV and can provide evidence of vaccine-induced immunity. However, the inherent heterogeneity in nasal antibody levels and variability in nasal specimen collection hinders the precise evaluation of mucosal antibody responses. Other studies have demonstrated that LAIV-induced immunity is also partially explained by T-cell immunity, serum antibody responses, and innate immunity, consistent with the multi-faceted nature of immunity induced by wild-type influenza infection and other live virus vaccines.     

Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: In vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006–2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable.