Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates

The development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. In this study, we compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67 ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus had glutamine and glycine at HA amino acid positions 226 and 228 (Q–G) that binds to α2-3 linked sialic acids, and one had leucine and serine that binds to α2-3 and α2-6 linked residues (L–S). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca Q–G and TEC67 ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67 ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57 ca virus from α2-3 to both α2-3 and α2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. We suggest that CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67 ca virus may be the most promising H2N2 LAIV candidate for further evaluation.     

Immunogenicity and protective efficacy of a live attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets

A novel 2009 influenza A (H1N1) virus was transmitted from humans to humans worldwide. The live attenuated monovalent A H1N1 vaccine (LAMV) for intranasal administration has shown promising immunogenicity and safety in clinical trials and for human use, but the experimental data based on LAMV is incomplete. In this study, using reverse genetic technology, we produced a cold-adapted (ca), live attenuated BJ/AA ca that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2009 pandemic A H1N1 isolate, A/Beijing/501/2009 virus (BJ501), and the remaining six internal gene segments from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). BJ/AA ca exhibited phenotypes of temperature sensitivity (ts), ca, and attenuation (att). The candidate BJ/AA ca was immunogenic in mice and induced strong mucosal secretory IgA (sIgA) in the respiratory tract. Two dosages of intranasal immunization induced robust HI antibodies and offered efficient protection against challenge by the wild-type (wt) 2009 pandemic A H1N1 (A/Beijing/501/2009 or A/California/07/2009) in mice and ferrets. These results support the evaluation of this vaccine made from a wt strain isolated in China for clinical trials.     

Replication of live attenuated influenza vaccine viruses in human nasal epithelial cells is associated with H1N1 vaccine effectiveness

In the 2013–2014 and 2015–2016 influenza seasons, live attenuated influenza vaccine (LAIV) generated reduced vaccine effectiveness (VE) against circulating H1N1 strains. This reduced VE coincided with the introduction of pandemic 2009 H1N1 (A/H1N1pdm09) vaccine virus reassortants, in place of pre-2009 seasonal H1N1 strains. Here, we explored one specific hypothesis for reduced VE; decreased replicative fitness of A/H1N1pdm09 strains in humans. Two A/H1N1pdm09 strains with reduced VE, A/California/07/2009 (A/CA09) and A/Bolivia/559/2013 (A/BOL13), were compared to pre-2009 seasonal H1N1 strains, A/New Caledonia/20/1999 (A/NC99) and A/South Dakota/6/2007 (A/SD07). Initial results showed that A/H1N1pdm09 strains had reduced multi-cycle infectivity in Madin-Darby Canine Kidney (MDCK) cells, compared to their pre-2009 counterparts. The A/BOL13 viral titre was found to be 2.65 log₁₀/mL lower when measured by multi-cycle 50% tissue culture infectious dose (TCID₅₀) assay compared to single-cycle fluorescent focus assay (FFA). By contrast, clinically effective A/NC99 titres differed by only 0.54 log₁₀/mL. In human alveolar (A549) cells, A/H1N1pdm09 strains replicated less than pre-2009 strains, with A/CA09 and A/BOL13 generating lower peak viral titres over 5 days. This phenotype was corroborated in physiologically relevant, primary human nasal epithelial cells (hNECs). Here, peak titres for pre-2009 strains A/NC99 and A/SD07 were 8.43 log₁₀ TCID₅₀/mL and 8.52 log₁₀ TCID₅₀/mL, respectively, versus 6.89 log₁₀ TCID₅₀/mL and 6.06 log₁₀ TCID₅₀/mL for A/H1N1pdm09 strains A/CA09 and A/BOL13. This confirmed a reduced ability of A/H1N1pdm09 strains to sustain replication in human respiratory cells. Using this information, H1N1 candidate A/Slovenia/2903/2015 (A/SLOV15) was characterised for replacement of A/BOL13 in the 2017/18 LAIV. A/SLOV15 produced comparable single and multi-cycle infectivity titres (Δ 0.16 log₁₀/mL) and reached a peak titre 1.23 log₁₀ TCID₅₀/mL higher than that of A/BOL13 in hNEC cultures. Taken together, these data suggest a reduction in sustained multi-cycle replication in human cells as a plausible root cause for reduced A/H1N1pdm09 VE.     

Generation of recombinant pandemic H1N1 influenza virus with the HA cleavable by bromelain and identification of the residues influencing HA bromelain cleavage

The proteolytic enzyme bromelain has been traditionally used to cleave the hemagglutinin (HA) protein at the C-terminus of the HA2 region to release the HA proteins from influenza virions. The bromelain cleaved HA (BHA) has been routinely used as an antigen to generate antiserum that is essential for influenza vaccine product release. The HA of the 2009 pandemic H1N1 influenza A/California/7/2009 (CA09) virus could not be cleaved efficiently by bromelain. To ensure timely delivery of BHA for antiserum production, we generated a chimeric virus that contained the HA1 region from CA09 and the HA2 region from the seasonal H1N1 A/South Dakota/6/2007 (SD07) virus that is cleavable by bromelain. The BHA from this chimeric virus was antigenically identical to CA09 and induced high levels of HA-specific antibodies and protected ferrets from wild-type H1N1 CA09 virus challenge. To determine the molecular basis of inefficient cleavage of CA09 HA by bromelain, the amino acids that differed between the HA2 of CA09 and SD07 were introduced into recombinant CA09 virus to assess their effect on bromelain cleavage. The D373N or E374G substitution in the HA2 stalk region of CA09 HA enabled efficient cleavage of CA09 HA by bromelain. Sequence analysis of the pandemic H1N1-like viruses isolated from 2010 revealed emergence of the E374K change. We found that K374 enabled the HA to be cleaved by bromelain and confirmed that the 374 residue is critical for HA bromelain cleavage.     

Efficacy of an AS03A-adjuvanted split H5N1 influenza vaccine against an antigenically distinct low pathogenic H5N1 virus in pigs

We used the pig model of influenza to examine the efficacy of an AS03(A)-adjuvanted split H5N1 (A/Indonesia/05/2005) vaccine against challenge with a low pathogenic (LP) H5N1 avian influenza (AI) virus (duck/Minnesota/1525/1981) with only 85% amino acid homology in its HA1. Influenza seronegative pigs were vaccinated twice intramuscularly with adjuvanted vaccine at 3 antigen doses, unadjuvanted vaccine or placebo. All pigs were challenged 4 weeks after the second vaccination and euthanized 2 days later. After 2 vaccinations, all pigs in the adjuvanted vaccine groups had high hemagglutination inhibiting (HI) antibody titers to the vaccine strain (160-640), and lower antibody titers to the A/Vietnam/1194/04 H5N1 strain and to 2 LP H5 viruses with 90-91% amino acid homology to the vaccine strain (20-160). Eight out of 12 pigs had HI titers (10-20) to the challenge virus immediately before challenge. Neuraminidase inhibiting antibodies to the challenge virus were detected in most pigs (7/12) and virus neutralizing antibodies in all pigs. There was no antigen-dose dependent effect on the antibody response among the pigs immunized with adjuvanted H5N1 vaccines. After challenge, these pigs showed a complete clinical protection, reduced lung lesions and a significant protection against virus replication in the respiratory tract. Though the challenge virus showed only moderate replication efficiency in pigs, our study suggests that AS03(A)-adjuvanted H5N1 vaccine may confer a broader protection than generally assumed. The pros and cons of the pig as an H5N1 challenge model are also discussed.     

Protective efficacy of a polyvalent influenza A DNA vaccine against both homologous (H1N1pdm09) and heterologous (H5N1) challenge in the ferret model

This study describes the protective efficacy of a novel influenza plasmid DNA vaccine in the ferret challenge model. The rationally designed polyvalent influenza DNA vaccine encodes haemagglutinin and neuraminidase proteins derived from less glycosylated pandemic H1N1 (2009) and H3N2 (1968) virus strains as well as the nucleoprotein (NP) and matrix proteins (M1 and M2) from a different pandemic H1N1 (1918) strain. Needle-free intradermal immunisation with the influenza DNA vaccine protected ferrets against homologous challenge with an H1N1pdm09 virus strain, demonstrated by restriction of viral replication to the upper respiratory tract and reduced duration of viral shedding post-challenge. Breadth of protection was demonstrated in two heterologous efficacy experiments in which animals immunised with the influenza DNA vaccine were protected against challenge with a highly pathogenic avian influenza H5N1 virus strain with reproducible survival and clinical outcomes.     

A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys

Human metapneumovirus (hMPV) infection causes respiratory tract disease similar to that observed during human respiratory syncytial virus infection (hRSV). hMPV infections have been reported across the entire age spectrum although the most severe disease occurs in young children. No vaccines, chemotherapeutics or antibodies are presently available for preventing or treating hMPV infections. In this study, a bovine/human chimeric parainfluenza virus type 3 (b/h PIV3) expressing the human parainfluenza type 3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins was engineered to express hMPV fusion (F) protein from the second genome position (b/h PIV3/hMPV F2) with the goal of generating a novel hMPV vaccine. b/h PIV3/hMPV F2 was previously shown to protect hamsters from challenge with wt hMPV (Tang RS, Schickli JH, Macphail M, Fernandes F, Bicha L, Spaete J, et al. Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity. J Virol 2003;77:10819-28) and is here further evaluated for efficacy and immunogenicity in African green monkeys (AGMs). AGMs immunized intranasally and intratracheally with b/h PIV3/hMPV F2 generated hMPV- and hPIV3-specific humoral and cellular immune responses and were protected from wt hMPV infection. In a separate study, the host-range restriction of b/h PIV3/hMPV F2 replication relative to wt hPIV3 was performed in rhesus monkeys to demonstrate attenuation. These studies showed that b/h PIV3/hMPV F2 was immunogenic, protective and attenuated in non-human primates and warrants further evaluation in humans as a vaccine candidate for prevention of hMPV-associated respiratory tract diseases.     

Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates

We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2–7 days post-challenge. All naïve macaques had detectable viral RNA from day 2–10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10–30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further development of a tetravalent EDIII-E2 dengue vaccine.     

Inactivated whole influenza virus particle vaccines induce neutralizing antibodies with an increase in immunoglobulin gene subclones of B-lymphocytes in cynomolgus macaques

The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or β-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes.     

An open label Phase I trial of a live attenuated H6N1 influenza virus vaccine in healthy adults

Background

We describe the results of an open label Phase I trial of a live attenuated H6N1 influenza virus vaccine (ClinicalTrials.gov Identifier: NCT00734175).

Methods and findings

We evaluated the safety, infectivity, and immunogenicity of two doses of 10⁷ TCID₅₀ of the H6N1 Teal HK 97/AA ca vaccine, a cold-adapted and temperature sensitive live, attenuated influenza vaccine (LAIV) in healthy seronegative adults.Twenty-two participants received the first dose of the vaccine, and 18 received the second dose of vaccine 4 weeks later. The vaccine had a safety profile similar to that of other investigational LAIVs bearing avian hemagglutinin (HA) and neuraminidase (NA) genes. The vaccine was highly restricted in replication: two participants had virus detectable by rRT-PCR beyond day 1 after each dose. Antibody responses to the vaccine were also restricted: 43% of participants developed a serum antibody response as measured by any assay: 5% by hemagglutination-inhibition assay, 5% by microneutralization assay, 29% by ELISA for H6 HA-specific IgG and 24% by ELISA for H6 HA specific IgA after either 1 or 2 doses. Following the second dose, vaccine specific IgG and IgA secreting cells as measured by ELISPOT increased from a mean of 0.6 to 9.2/10⁶ PBMCs and from 0.2 to 2.2/10⁶ PBMCs, respectively.

Conclusion

The H6N1 LAIV had a safety profile similar to that of LAIV bearing other HA and NA genes, but was highly restricted in replication in healthy seronegative adults. The H6N1 LAIV was also not as immunogenic as the seasonal LAIV.     

Introducing point and deletion mutations into the P/C gene of human parainfluenza virus type 1 (HPIV1) by reverse genetics generates attenuated and efficacious vaccine candidates

The P/C gene of human parainfluenza virus type 1 (HPIV1) encodes a nested set of related accessory C proteins, C'/C/Y1/Y2, which have been shown in other paramyxoviruses to have a role in evasion of the type I interferon (IFN) response following virus infection. We previously demonstrated that a set of two amino acid substitutions, CR84G/HNT553A, and a separate amino acid substitution, CF170S, are independently attenuating for HPIV1 in African green monkeys (AGMs). However, in each case the attenuation (att) phenotype is vulnerable to reversion by a single nucleotide change back to wild type. Using reverse genetics, recombinant HPIV1 (rHPIV1) vaccine candidates were generated that were designed for increased genetic and phenotypic stability by: (i) creating a two-amino acid deletion and substitution at the site of the CF170S mutation, yielding CDelta170; (ii) introducing a six amino acid deletion in the N-terminal region of C, CDelta10-15; and (iii) combining these stable deletion mutations with the att CR84G/HNT553A mutation. The resulting rHPIV1 vaccine candidates were evaluated for attenuation in hamsters and AGMs and for immunogenicity and protective efficacy in AGMs. The CDelta10-15 mutation was attenuating in hamsters but not in AGMs, and likely will be of limited value for an HPIV1 vaccine. Conversely, the CR84G/HNT553A mutation set was attenuating in AGMs but not in hamsters. Thus, these two mutations demonstrated reciprocal host range phenotypes involving different regions of C. The CDelta170 mutation conferred a significant level of attenuation in hamsters and AGMs that closely resembled that of CF170S and will be of particular utility for vaccine development because it involves a deletion of six nucleotides rendering it highly refractory to reversion. The combination of the CR84G/HNT553A mutation set and the CDelta170 deletion mutation yielded a virus, rCR84G/Delta170 HNT553A, that exhibited a satisfactory level of attenuation in hamsters and AGMs and was immunogenic and highly protective against HPIV1 wt challenge. This virus will be evaluated clinically as a live intranasal HPIV1 vaccine, one that can be further attenuated as necessary by the introduction of additional stabilized att mutations previously developed in the L protein.     

Serological evaluation of an influenza A virus cold-adapted reassortant live vaccine, CR-37 (H1N1), in Japanese adult volunteers.

A cold-adapted influenza A virus, CR-37 (H1N1), derived from genetic reassortment between A/Ann Arbor/6/60 (H2N2) cold-adapted variant virus and A/California/10/78 (H1N1) wild-type virus, was tested in Japanese adult volunteer. The CR-37 live virus preparation induced only low-grade clinical reactions in volunteers for the first 3-4 days after inoculation. Two vaccinees who did not show any antibody changes became febrile (over 38.0 degrees C). Skin tests using the vaccine preparation and uninfected allantoic fluid were performed, and indicated that one of these two vaccines was positive for the CR-37 vaccine preparation. A high proportion of the vaccinees whose sera had a haemagglutination-inhibition (HI) antibody titre against the vaccine strain of less than or equal to 64 before inoculation, seroconverted in both HI and neuraminidase-inhibition (NAI) antibody titrations, and only a few seroconverted in the titration of antibody against type-specific internal antigens. The serological examinations against heterotypic H1N1 variants indicated that the cold-adapted live influenza virus vaccine could induce a broad spectrum of HI antibody reactivity and immunity of long duration.     

Serological evaluation of an influenza A virus cold-adapted reassortant live vaccine, CR-37 (H1N1), in Japanese adult volunteers

A cold-adapted influenza A virus, CR-37 (H1N1), derived from genetic reassortment between A/Ann Arbor/6/60 (H2N2) cold-adapted variant virus and A/California/10/78 (H1N1) wild-type virus, was tested in Japanese adult volunteer. The CR-37 live virus preparation induced only low-grade clinical reactions in volunteers for the first 3-4 days after inoculation. Two vaccinees who did not show any antibody changes became febrile (over 38.0 degrees C). Skin tests using the vaccine preparation and uninfected allantoic fluid were performed, and indicated that one of these two vaccines was positive for the CR-37 vaccine preparation. A high proportion of the vaccinees whose sera had a haemagglutination-inhibition (HI) antibody titre against the vaccine strain of less than or equal to 64 before inoculation, seroconverted in both HI and neuraminidase-inhibition (NAI) antibody titrations, and only a few seroconverted in the titration of antibody against type-specific internal antigens. The serological examinations against heterotypic H1N1 variants indicated that the cold-adapted live influenza virus vaccine could induce a broad spectrum of HI antibody reactivity and immunity of long duration.     

Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus

A human influenza A virus X-31 (high-yielding strain) was cold-adapted for possible future use as live attenuated vaccine. Mutant influenza viruses were selected during successive serial passage in embryonated hens' eggs at progressively lower sub-optimal temperature (30, 27 degrees C followed by 24 degrees C). The cold-passaged mutant exhibited both temperature-sensitivity (ts) and cold-adapted (ca) phenotypes. The pathogenicity and immunogenicity of X-31 ca virus were studied in mice following intranasal inoculation. The mice did not show clinical signs even at high titer infection. Immunization of mice with X-31 ca virus elicited high titers of neutralizing antibody and provided complete protection against homologous and heterologous virus challenges. To assess the genetic stability, the X-31 ca virus was passaged at 37 degrees C in MDCK cells or inoculated into mice. Revertant virus was not found in the lungs of any of the mice and the supernatants of the MDCK culture. We conclude that the X-31 ca candidate vaccine virus exhibits the desired level of attenuation, immunogenicity, and protective efficacy required for live attenuated vaccine and merits further evaluation at clinical level.     

Association between Haemagglutination inhibiting antibodies and protection against clade 6B viruses in 2013 and 2015

BackgroundThe epidemiology of the pandemic A(H1N1) virus has been changing as population immunity continues to co-evolve with the virus. The impact of genetic changes in the virus on human’s susceptibility is an outstanding important question in vaccine design. In a community-based study, we aim to (1) determine the genetic characteristics of 2009–2015 pandemic H1N1 viruses, (2) assess antibody response following natural infections and (3) assess the correlation of A/California/07/09 antibody titers to protection in the 2013 and 2015 epidemics.MethodsIn a household transmission study, serum specimens from 253 individuals in Managua, Nicaragua were analyzed. Combined nose and throat swabs were collected to detect RT-PCR confirmed influenza infection and virus sequencing. Hemagglutination inhibition assays were performed and the protective titer for circulating H1N1pdm was determined.ResultsClade 6B pandemic H1N1 viruses predominated in Nicaragua during the 2013 and 2015 seasons. Our household transmission study detected a household secondary attack rate of 17% in 2013 and 33% in 2015. Infected individuals, including vaccinees, showed an apparent antibody response to A/California/07/09. Baseline titers of A/California/07/09 antibodies were found to associate with protection in both seasons. A titer of ≥1:40 correlated to a 44% protection in children, a 29% protection in adults 15–49 years old and a 51% protection in adults 50–85 years old.ConclusionIn 2013 and 2015, antibody titers to A/California/07/09 associated with an infection risk reduction amongst exposed household contacts. This is consistent with a detectable vaccine effectiveness reported in a number of studies. Genetic changes in clade 6B viruses might have led to a reduced immunity in some whereas others might have been less affected. The use of human serologic data is important in virus characterization and if performed in a timely manner, could assist in vaccine strain selection.     

2018-2019年宁夏甲型H1N1流感病毒血凝素基因组序列分析

目的 了解宁夏2018-2019流感监测年度流感病毒病原学检测情况,分析甲型H1N1流感病毒血凝素（HA）基因特征。方法 采用real time RT-PCR方法对流感监测哨点医院采集的流感样病例（ILI）标本进行核酸检测;对阳性标本进行毒株分离;提取甲型H1N1毒株的RNA,采用RT-PCR方法扩增HA片段并测序,利用生物信息软件对测序结果进行比对分析。结果 宁夏流感网络实验室检测咽拭子标本共5214份,核酸检测阳性数为760份,其中甲型H1N1阳性数为485份,占总阳性数的63.82%,分离出甲型H1N1毒株 161株。宁夏分离毒株与疫苗株A/Califaoria/07/2009不在同一进化分支,同源性为92.6%~96.3%;与疫苗株A/Michigan/45/2015(H1N1)为同一进化分支,同源性为96.6%~98.1%。与疫苗株A/Califaoria/07/2009比较,抗原位点、受体结合位点及其他位点均有变异,除毒株 A/Ningxia_Xixia/SWL1176/2019(H1N1)第222位氨基酸发生D222G变异外,其他甲型H1N1流感毒株均未发生D222G变异。所有毒株增加糖基化位点162NQT,个别毒株糖基化位点增加2~3个。结论 宁夏2018 -2019年度流感优势毒株为甲型H1N1毒株。序列分析表明甲型H1N1病毒发生了不同程度的变异,在抗原特异性、毒力和感染性上有可能已经发生变化,需要及时更换疫苗株成分。     

Efficacy of SAG-2 oral rabies vaccine in two species of jackal (Canis adustus and Canis mesomelas)

Trials were carried out to test the efficacy of SAG-2 oral rabies vaccine in two species of jackals, namely the side-striped jackal (C. adustus) and the black-backed jackal (C. mesomelas). The first trial tested the efficacy of SAG-2 when given by direct oral administration at doses of 6.5 and 7.5 log10 median tissue culture infectious doses (TCID50). One side-striped jackal which had received the higher dose did not seroconvert and succumbed to challenge, while all other jackals seroconverted and resisted a lethal challenge. The second trial tested the efficacy in side-striped jackals only of the SAG-2 vaccine when given within chicken head baits. A volume of 1.8 ml of vaccine fluid with titers of 7.0 or 8.0 log10 TCID50/ml were placed into blisters which were stapled under the skin of the chicken heads. All jackals (5/5) which received 8.0 log10 TCID50/ml and 3 of 5 which received 7.0 log10 TCID50/ml seroconverted and resisted lethal challenge. A third trial tested the rate of vaccine virus titer loss in chicken head baits placed under field conditions. Titer loss was marked in baits which were not protected from direct sunshine, whereas under vegetation cover approximately log10 TCID50/ml was lost every 3 days. Hence, it was concluded that a bait vaccine virus titer of 8.0 log10 TCID50/ml will be sufficient to immunize wild jackal populations if enough baits can be consumed by jackals within 3 days. This conclusion needs to be tested through the use of oral vaccine in field trials.     

Compatibility of ASO3-adjuvanted H1N1pdm09 and seasonal trivalent influenza vaccines in adults: results of a randomized, controlled trial

When Canada chose a novel adjuvanted vaccine to combat the 2009 influenza pandemic, seasonal trivalent inactivated vaccine (TIV) was also available but compatibility of the two had not been assessed. To compare responses after concurrent or sequential administration of these vaccines, adults 20-59 years old were randomly assigned (1:1) to receive ASO3-adjuvanted H1N1pdm09 vaccine (Arepanrix, GSK, Quebec City, Quebec), with TIV (Vaxigrip, Sanofi Pasteur, Toronto) given concurrently or 21 days later. Blood was obtained at baseline and 21 days after each vaccination to measure hemagglutination inhibition (HAI) titers. Adverse effects were assessed using symptom diaries and personal interviews. 282 participants completed the study (concurrent vaccines 145, sequential vaccines 137). HAI titers to H1N1pdm09 were ≥ 40 at baseline in 15-18% of participants and following vaccination in 91-92%. Initially seropositive subjects (titer ≥ 10) had lower H1N1pdm09 geometric mean HAI titers (GMT) after concurrent than separate vaccinations (320.0 vs 476.5, p=0.039) but both exceeded GM responses of initially na?ve participants, which were unaffected by concurrent TIV. Responses to TIV were not lower after concurrent than separate vaccination. Adverse event rates were not increased by concurrent vaccinations above those with H1N1pdm09 vaccine alone. This adjuvanted H1N1pdm09 vaccine was immunogenic and compatible with concurrently administered TIV.     

Evaluation in children of cold-adapted influenza B live attenuated intranasal vaccine prepared by reassortment between wild-type B/Ann Arbor/1/86 and cold-adapted B/Leningrad/14/55 viruses

A reassortant cold-adapted (ca) influenza B experimental live attenuated intranasal vaccine was evaluated for safety and immunogenicity in children by means of a blind, placebo controlled study. The vaccine contained the haemagglutinin and neuraminidase genes, and the gene for its non-structural proteins from wild-type (wt) B/Ann Arbor/1/86 virus, the contemporary strain at the time of the study. Other genes were derived from ca B/Leningrad/14/55 virus. No increase in illness rates was seen in the children from ages 3-15 years given vaccine at maximum potency (a one in two dilution of infectious allantoic fluid, having a titre of 10(7.0) EID50) compared to children given placebo. About 60% of seronegative children, ages 3-7 years, exhibited a detectible antibody response following one dose of intranasal vaccine, with the seroresponse rate rising to greater than 70% after two doses of vaccine. Immunogenicity was lowest in seropositive children age 8-15 years, reaching a maximum of 36% after two doses. Results indicated that the vaccine was highly attenuated, and probably of adequate immunogenicity for kindergarten age children. The lower immunogenicity in older children suggests the vaccine might be overly attenuated for use in school-age children who are more likely to have a history of prior natural infection with influenza B virus. Further clinical and epidemiological studies of protection are needed to fully assess this.