Reassortant Vaccine Viruses Research Articles

Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model

This study sought to improve an existing live attenuated influenza vaccine (LAIV) by including nucleoprotein (NP) from wild-type virus rather than master donor virus (MDV). H7N9 LAIV reassortants with 6:2 (NP from MDV) and 5:3 (NP from wild-type virus) genome compositions were compared with regard to their growth characteristics, induction of humoral and cellular immune responses in mice, and ability to protect mice against homologous and heterologous challenge viruses. Although, in general, the 6:2 reassortant induced greater cell-mediated immunity in C57BL6 mice than the 5:3 vaccine, mice immunized with the 5:3 LAIV were better protected against heterologous challenge. The 5:3 LAIV-induced CTLs also had better in vivo killing activity against target cells loaded with the NP366 epitope of recent influenza viruses. Modification of the genome of reassortant vaccine viruses by incorporating the NP gene from wild-type viruses represents a simple strategy to improve the immunogenicity and cross-protection of influenza vaccines.

Development of an Influenza A Master Virus for Generating High-Growth Reassortants for A/Anhui/1/2013(H7N9) Vaccine Production in Qualified MDCK Cells.

In 2013, the first case of human infection with an avian influenza A virus (H7N9) was reported in China, and the human infection with this virus has continued as of 2016. At the request of the WHO, we have successfully developed candidate reassortant vaccine virus using A/Anhui/1/2013 and the high egg-growth master virus A/PR/8/1934. Recent plans regarding influenza vaccine production include using cell-cultured systems in Japan and several other countries. However, egg-based vaccine viruses are not always suitable for cell-cultured vaccine production due to potential issues with growth, protein yield and antigenic stability. Therefore, in this study, we have developed a high-growth master virus (hg-PR8) adapted to qualified NIID-MDCK cells that are competent for vaccine production. The virus hg-PR8 was obtained after 20 serial passages of A/Puerto Rico/8/1934 (PR8) in NIID-MDCK cells. The viral titer of hg-PR8 was 108.6 plaque-forming units per milliliter (PFU/mL). Seven amino acid substitutions were identified in the amino acid sequences of PB2, PB1, PA, NA, M and NS of hg-PR8 compared to the sequence of the original PR8 (org-PR8) strain. The growth capacities of the reassortant viruses, which possess heterologous internal genes from hg-PR8 or org-PR8, indicated that the amino acid changes in PB2 and NS2 similarly affected growth capacity in NIID-MDCK cells. To assess the suitability of hg-PR8 as a master virus, we generated 6:2 reassortant viruses possessing the HA and NA segments from A/Anhui/1/2013 (H7N9) and the remaining segments from hg-PR8. The virus titers of the reassortant strains were 107−108 PFU/mL. The antigenicity of the viruses was stable during ten passages of the viruses in NIID-MDCK cells. In comparison with the egg-based reassortant vaccine viruses with identical HA and NA segments, the hg-PR8-based viruses showed 1.5- to 2-fold higher protein yields in NIID-MDCK cells.

Evaluation of MDCK cell-derived influenza H7N9 vaccine candidates in ferrets.

Avian-origin influenza A (H7N9) viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300μg aluminum hydroxide, 1.5μg HA, and 1.5μg HA plus 300μg aluminum hydroxide) were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA) inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only), 17 and 80 for the unadjuvanted (HA only) group, and 190 and 640 for the adjuvanted group (HA plus adjuvant), respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted.

Seroconversion of 2009 pandemic influenza A (H1N1) vaccination in kidney transplant patients and the influence of different risk factors

Influenza may present a high morbidity and mortality in solid organ transplanted patients (SOTP). Annual influenza virus vaccine is recommended for SOTP. However, low levels of seroconversion in SOTP have been reported. The aim of this study was to evaluate the immunogenicity of 2009 pandemic influenza A (H1N1) - A(H1N1)pdm09--vaccine in kidney transplant patients and to analyze which features might affect seroconversion. This study was conducted from March to August 2010 at the Renal Transplantation Unit of University of São Paulo, Brazil. A total of 85 renal transplant patients attending the outpatient unit received one 15-μg intramuscular dose of A(H1N1) pdm09 influenza vaccine (reassortant vaccine virus A/California/7/2009 [NYMC X-179A]). Blood samples were collected immediately before and 21 days after the vaccine was given. Antibody response was measured by the standard hemagglutination-inhibition (HI) assay. The primary immunogenicity endpoint for this study was seroconversion in previously seronegative patients (HI titers <1:40), and the secondary endpoint was the identification of features that could affect seroconversion in this population. Five (5.9%) patients presented HI titers prevaccination ≥ 1:40 and were excluded from further analysis. Seroconversion in previously negative patients occurred in 27 (34%) of 80 patients. Prevaccination HI titers geometrical mean was 5.8 and postvaccination 19.6 (ratio 3.4). Significant seroconversion rate factors were female gender, non-Caucasian ethnicity, and post-transplant time before vaccination. No impact was seen on seroconversion for age, donor type, tacrolimus and cyclosporine blood levels, renal function, or blood lymphocyte counts. Mycophenolate (MPA) showed a lower rate of seroconversion when compared with azathioprine. Tacrolimus and cyclosporine had similar seroconversion rates. Sirolimus use was associated with the highest rate of seroconversion, although these patient numbers were low. Immunosuppresssion containing MPA was considerably less effective in seroconversion than drug combinations with no MPA. Patients receiving sirolimus had more chance of seroconversion. HI titers geometric means pre/post vaccine were as follows: MPA (n = 56): 5.8/12.8; tacrolimus (n = 50): 5.9/16.2; cyclosporine (n = 18): 5.4/24.2; azathioprine (n = 19): 6.2/51.6; and sirolimus (n = 6): 8/80. By univariate analysis, being female and non-White were variables associated with 3.3 times more chance of seroconversion than being male and White. In the multivariate analysis, the variables remaining in the model showed similar hazard ratios. In this study, the monovalent A(H1N1)pdm09 influenza vaccine demonstrated low rates of seroconversion, particularly in patients on MPA, but with potentially higher response rates in patients on sirolimus.

Identification of Critical Residues in the Hemagglutinin and Neuraminidase of Influenza Virus H1N1pdm for Vaccine Virus Replication in Embryonated Chicken Eggs

In 2009, we successfully produced a high-yield live attenuated H1N1pdm A/California/7/2009 vaccine (CA/09 LAIV) by substitution of three residues (K119E, A186D, and D222G) in the hemagglutinin (HA) protein. Since then, we have generated and evaluated additional H1N1pdm vaccine candidates from viruses isolated in 2010 and 2011. The 2010 strains with the new HA substitutions near the HA receptor binding site (N125D and D127E or D127E and K209E) grew well in eggs and formed large plaques in Madin-Darby canine kidney (MDCK) cells. Introduction of these acidic amino acids into the HA of CA/09 also improved vaccine virus growth in eggs to a titer comparable to that of CA/09 LAIV. However, the high growth of A/Gilroy/231/2011 (Gil/11) vaccine virus required modification in both the HA and the NA segments. The residue at position 369 of the NA was found to be critical for virus replication in MDCK cells and eggs. These HA and NA residues had minimal impact on viral entry but greatly improved viral release from infected cells. Our data implied that the HA receptor binding and NA receptor cleaving function of the poor-growth H1N1pdm virus was not well balanced for virus replication in host cells. The high-growth vaccine candidates described in this study maintained vaccine virus antigenicity and induced high levels of neutralizing antibodies in immunized ferrets, making them suitable for vaccine production. The identification of the amino acids and their roles in viral replication should greatly help vaccine manufacturers to produce high-yield reassortant vaccine viruses against the future drifted H1N1pdm viruses.

Adaptation of High-Growth Influenza H5N1 Vaccine Virus in Vero Cells: Implications for Pandemic Preparedness

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 108 TCID50/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.

Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk

The H5N1 subtype of highly pathogenic avian influenza viruses has spread to over 63 countries in Asia, Europe, and Africa and has become endemic in poultry. Since 2004, vaccination against H5N1 influenza has become common in domestic poultry operations in China. Most influenza vaccines have been produced in embryonated chicken eggs. High yield is the essential feature of a good vaccine candidate virus. Therefore, the large-scale manufacture of such a vaccine requires that the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be increased. We generated two sets of reassortant H5N1 viruses based on backbone viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse genetics. The HAs and NAs of the reassortants were derived from the three epidemic H5N1 strains found in China. We compared the replication properties of these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after inserting either 20 or 38 amino acids into their NA stalks. In this study, we demonstrated that inserting 38 amino acids into the NA stalks can significantly increase the viral yield of H5N1 reassortant viruses in both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids into the same NA stalks does not. Hemagglutinin inhibition testing and protection assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA stalks had the same antigenicity as the viruses with wt-NA. These results suggest that the generation of an H5N1 recombinant vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and more economical strategy for the increase in viral yield in both eggs and MDCK cells for the purposes of vaccine production.

Reverse genetic platform for inactivated and live-attenuated influenza vaccine

Influenza vaccine strains have been traditionally developed by annual reassortment between vaccine donor strain and the epidemic virulent strains. The classical method requires screening and genotyping of the vaccine strain among various reassortant viruses, which are usually laborious and time-consuming. Here we developed an efficient reverse genetic system to generate the 6:2 reassortant vaccine virus from cDNAs derived from the influenza RNAs. Thus, cDNAs of the two RNAs coding for surface antigens, haemagglutinin and neuraminidase from the epidemic virus and the 6 internal genes from the donor strain were transfected into cells and the infectious viruses of 6:2 defined RNA ratio were rescued. X-31 virus (a high- growth virus in embryonated eggs) and its cold-adapted strain X-31 ca were judiciously chosen as donor strains for the generation of inactivated vaccine and live-attenuated vaccine, respectively. The growth properties of these recombinant viruses in embryonated chicken eggs and MDCK cell were indistinguishable as compared to those generated by classical reassortment process. Based on the reverse genetic system, we generated 6+2 reassortant avian influenza vaccine strains corresponding to the A/Chicken/Korea/ MS96 (H9N2) and A/Indonesia/5/2005 (H5N1). The results would serve as technical platform for the generation of both injectable inactivated vaccine and the nasal spray live attenuated vaccine for the prevention of influenza epidemics and pandemics.

The effect of influenza B virus M segment on the replication of reassortant vaccine viruses

Flumist™ is a cold-adapted (ca), live attenuated influenza virus vaccine containing H1N1, H3N2, and B strains. The vaccine strains have a 6:2 gene constellation. Six gene segments, PB1, PB2, PA, NP, M and NS, are derived from the master donor viruses, ca A/Ann Arbor/6/60(MDV-A) and ca B/Ann Arbor/1/66(MDV-B), and two gene segments, the hemagglutinin (HA) and neuraminidase (NA), are derived from circulating wild-type strains. We observed that the M gene segment of influenza B virus reassortants can influence the replication of these viruses in CEK cells. Replication curves revealed that a 6:2 reassortant B virus (wt HA and wt NA) replicated less well, by approximately 2 log 10, than the corresponding 5:3 strain (wt HA, wt NA and wt M) at 33 °C in chicken embryo kidney (CEK) cells; this difference was not observed at 25 °C.

Safety evaluation in chickens of candidate human vaccines against potential pandemic strains of influenza.

Two candidate formalin-inactivated vaccines, made from high-growth reassortant viruses with the HA and NA genes from avian viruses in a background of genes derived from A/Puerto Rico/8/34 (PR8), were prepared against H5N1 and H9N2 subtypes (designated as H5N1/PR8 and H9N2/PR8, respectively). These viruses bear the genotypes, antigenicity, and attenuation in mouse models that are desirable in candidate vaccines. The pathogenicity of the newly generated avian-human reassortant vaccine viruses was also evaluated in chickens. Neither H5N1/PR8 nor H9N2/PR8 were highly pathogenic for chickens. No clinical signs, gross legions, or histological lesions were observed in chickens that were administered H5N1/PR8 either intranasally (i.n.) or intravenously (i.v.), and virus was not detected in oropharyngeal or cloacal swabs. When H9N2/PR8 was administered i.n., no clinical signs, gross lesions, or histological lesions were observed and no virus was detected in cloacal swabs. However, virus was isolated at low titer from oropharyngeal swabs of all eight chickens. Although no clinical signs were observed when H9N2/PR8 was administered i.v., mild tracheitis was seen in one of two chickens. Moderate amounts of antigen were observed in tracheal respiratory epithelium, and low titers of virus were recovered from oropharyngeal and cloacal swabs of some chickens. In summary, both reassortant vaccine viruses replicated poorly in chickens. These studies suggest that these candidate vaccine viruses carry a low risk of transmission to chickens.

Are there alternative avian influenza viruses for generation of stable attenuated avian-human influenza A reassortant viruses?

The present study evaluated gull influenza A viruses as donors of attenuating genes for the production of live, attenuated influenza A H1N1 and H3N2 avian-human (ah) reassortant viruses for use as vaccines to prevent disease due to influenza A viruses in humans. The previously evaluated duck influenza A viruses were abandoned as donors of attenuating avian influenza virus genes because clinical evaluation of H1N1 and H3N2 ah reassortant virus vaccines derived from duck viruses documented residual virulence of H1N1 reassortants for seronegative infants and young children. Gull influenza A viruses occupy an independent ecologic niche and are rarely isolated from species other than gulls. The possibility of using gull influenza A viruses as donors of internal gene segments in ah reassortant viruses was evaluated in the present study using three different gull viruses and three human influenza A viruses. Gull-human H3N2 reassortant influenza A viruses with the desired 6-2 genotype (six internal avian influenza virus genes and the two human influenza virus surface glycoprotein genes) were readily generated and were found to be attenuated for squirrel monkeys and chimpanzees. However, ah reassortant viruses with gull and human influenza A H1N1 genes were difficult to generate, and reassortants that had the desired genotype of six gull virus genes with human influenza A H1 and N1 genes were not isolated despite repeated attempts. The gull PB2, NP and NS genes were not present in any of the gull-human H1N1 reassortants generated. The under-representation of these three gene segments suggests that reassortants bearing one or more of these three gene segments might have reduced viability indicative of a functional incompatibility in their gene products. The difficulties encountered in the generation of a 6-2 gull-human H1N1 reassortant virus are sufficient to conclude that the gull influenza A viruses tested would not be useful as donors of sets of six internal genes to attenuate human influenza A viruses. This study also identifies influenza virus gene segments that appear to be incompatible for generation of reassortants. Elucidation of the molecular basis of this restriction may provide information on intergenic interactions involved in virion assembly or packaging.

Nucleotide sequence changes in the polymerase basic protein 2 gene of temperature-sensitive mutants of influenza A virus

Influenza A viruses bearing temperature-sensitive (ts) mutations are restricted in replication in the respiratory tract of animals and humans and are therefore attenuated. Nucleotide sequences were determined for the RNA segment coding for the polymerase basic protein 2 (PB2) from a panel of 12 influenza A/Udorn/307/72 (H3N2) is viruses, previously characterized to have a is mutation in the PB2 gene. Each of the viruses with a is mutation in the PB2 gene had a single amino acid change located at position 65, 100, 112, 174, 298, 310, 386, 391, 556, or 658 of the PB2 protein. The sites of the single mutations were scattered throughout the length of the protein and occurred in regions that are highly conserved among the influenza A virus PB2 predicted amino acid sequences. Interestingly, the substitution of aspartic acid for asparagine at position 556 was found to lie within a region that has homology with cap-binding motifs of human and yeast proteins. Taken together, the findings of lesion sites in the A/Udorn/307/72 PB2 gene and the three reported amino acid changes at positions 265, 417, and 512 for A/AA/6/60, A/WSN/33, and A/FPV/Ros/34 is PB2 genes, respectively, indicate that the PB2 gene can sustain a viable is mutation at different sites. This information will allow us to construct cloned cDNA copies of the A/Udorn/307172 PB2 gene mutagenized at specific sites. Different configurations of two or more is mutations may be incorporated into the cDNA PB2 gene constructs. We have a host-range reassortant virus that should permit rescue of in vitro-produced transcripts of the PB2 gene into infectious virus. The rescue of these mutated PB2 RNA segments into an infectious influenza A virus may lead to the development of live attenuated reassortant virus vaccines that are satisfactorily attenuated, genetically stable, and immunogenic in humans.

Characterization of the attenuating M and NP gene segments of the avian influenza A/Mallard/78 virus during in vitro production of avian-human reassortant vaccine viruses and after replication in humans and primates

A unique requirement for live attenuated reassortant influenza vaccines is the need to generate new reassortant vaccine viruses with the appearance of each new antigenic variant. Thus, the attenuation phenotype conferred by the attenuated donor influenza virus must remain genetically stable during the generation of each new reassortant vaccine virus. In this study we used nucleotide sequence analysis to evaluate the genetic stability of the attenuating M and NP genes of the avian influenza A/Mallard/NY/6750/78 attenuated donor virus during the in vitro generation and subsequent in vivo replication of avian-human (AH) influenza A reassortant vaccine viruses in monkeys and humans. Nucleotide sequence changes in the M and NP genes occurred at a rate of ≈0.61 substitutions/1000 nt/reassortant during in vitro generation of four AH reassortant viruses. Only two nucleotide sequence changes occurred in the M and NP gene segments of four isolates of HINI or H3N2 AH vaccine viruses following 6–8 days of replication in seronegative children, and neither change affected amino acids previously identified as playing a potential role in attenuation. In addition, there were no changes in the nucleotide sequence of the M and NP genes of single gene AH reassortant viruses following five serial passages in squirrel monkeys. Finally, there was no change in the level or duration of replication of the single gene reassortant viruses in the upper or lower respiratory tract of monkeys following serial passage. These results suggest that nucleotide sequence changes occur infrequently during the production of reassortant influenza A vaccine viruses and their subsequent replication in humans and monkeys, and that genetic instability does not appear to represent a significant obstacle to this approach for production of live attenuated vaccines.

The A/Mallard/67S0/78 Avian-Human, but Not the A/Ann Arbor/6/60 Cold-Adapted, Influenza A/Kawasaki/86 (H1N1) Reassortant Virus Vaccine Retains Partial Virulence for Infants and Children

Characteristics of avian-human (ah) and cold-adapted (ca) influenza A/Kawasaki/9/86 (H1N1) reassortant vaccine viruses were compared in 37 seronegative adults and 122 seronegative infants and children. The 50% human infectious dose (HID50) in infants and children was 10(2.9) and 10(2.6) TCID50 for the ah and ca vaccine, respectively. The ah influenza A/Kawasaki/9/86 reassortant was reactogenic: 24% of infants and children infected with greater than or equal to 100 HID50 had fever greater than or equal to 39.4 degrees C. Since H3N2 ah vaccines were previously shown to be adequately attenuated, it is reasonable to suggest that the genes that code for hemagglutinin and neuraminidase of the H1N1 virus apparently influence the reactogenicity of reassortant viruses derived from the avian influenza A/Mallard/New York/6750/78 donor virus. Because this avian virus does not reproducibly confer a satisfactory level of attenuation to each subtype of influenza A virus, it is not a suitable donor virus for attenuation of wild-type influenza viruses. In contrast, the ca A/Ann Arbor/6/60 donor virus reliably confers attenuation characteristics to a variety of H1N1 and H3N2 influenza A viruses.

Safety and Immunogenicity of Live Attenuated Cold-Adapted Influenza BlAnn Arbor/1/86 Reassortant Virus Vaccine in Infants and Children

A cold-adapted (ca) influenza B reassortant vaccine consisting of two genes encoding the hemagglutinin and neuraminidase from wild-type influenza B/Ann Arbor/1/86 virus and the six internal RNA segments from influenza B/Ann Arbor/1/66 ca virus was evaluated in 18 seropositive and 57 seronegative infants and children. The ca reassortant was infectious in seronegative vaccinees, with an estimated 50% human infectious dose of 10(2.5) TCID50. Nasal wash specimens from vaccinees retained the temperature-sensitive phenotype, indicating that the virus was phenotypically stable after replication in fully susceptible children. The vaccine was highly immunogenic in the seronegative vaccinees; 54% of the seropositive vaccinees also developed an increase in serum antibody. The ca vaccine was well tolerated, with only a mild increase in upper respiratory tract symptoms seen in the seronegative vaccinees. These studies indicate that the B/Ann Arbor/1/86 ca reassortant is safe, immunogenic, and phenotypically stable in infants and children.

Comparison of Live Attenuated Cold-Adapted and Avian-Human Influenza A/Bethesda/85 (H3N2) Reassortant Virus Vaccines in Infants and Children

Randomized, placebo-controlled studies with 10(3)-10(7) 50% tissue-culture infectious dose (TCID50) of avian-human (ah) and cold-adapted (ca) influenza A/Bethesda/85 (H3N2) reassortant viruses were completed in 106 seronegative young children 6-48 months of age. Although the reassortants differed in six of eight RNA segments, they exhibited similar properties in level of attenuation, infectivity, immunogenicity, and efficacy. The 50% human infectious dose was 10(4.6) TCID50 for ah and 10(4.4) for ca vaccines. Both reassortants were satisfactorily attenuated with restricted replication and were no more reactogenic than placebo. The mean peak titer of virus shed was 10(1.5) (ah) to 10(2.0) (ca) TCID50/ml, and each of 37 isolates tested retained their characteristic vaccine phenotypes. Infection with ah or ca virus conferred immunity to experimental challenge with homologous virus. These findings indicate that both ah and ca influenza A/Bethesda/85 (H3N2) reassortants should be suitable vaccine candidates for use in healthy infants and young children.

Evaluation of the Infectivity, Immunogenicity, and Efficacy of Live Cold-Adapted Influenza B/Ann Arbor/1/86 Reassortant Virus Vaccine in Adult Volunteers

A cold-adapted (ca) influenza B reassortant that derived two genes encoding the hemagglutinin and neuraminidase from influenza B/Ann Arbor/1/86 wild-type virus and six internal RNA segments from ca influenza B/Ann Arbor/1/66 virus was evaluated in 66 adult volunteers having a serum hemagglutination inhibition antibody titer less than or equal to 1:8. The ca reassortant was attenuated and elicited the production of systemic and local antibodies; the 50% human infectious dose was 10(6.4) TCID50. Six weeks after vaccination, 12 unvaccinated volunteers and 13 recipients of ca virus (10(7.5) TCID50) were challenged experimentally with homologous wild-type influenza B virus. The ca vaccine completely protected against illness, and the magnitude of shedding was 50-fold less in vaccinees than in unimmunized controls, five of whom became ill. These findings indicate that the six internal RNA segments of the ca influenza B/Ann Arbor/66 donor virus confer desirable properties of a live virus vaccine to a reassortant derived from a virulent virus. Such reassortants may be suitable vaccines for healthy adults.

Nucleotide sequence of the avian influenza A/Mallard/NY/6750/78 virus polymerase genes

The avian influenza A/Mallard/NY/6750/78 virus is currently being evaluated as a donor of attenuating genes in the construction of live avian-human influenza A reassortant virus vaccines for use in humans. We determined the nucleotide sequences of the three polymerase gene segments of this virus. This completes the nucleotide sequence of the six transferrable genes of the avian donor virus. Comparison of the nucleotide and deduced amino acid sequences of the non-glycoprotein genes of the avian A/Mallard/78 virus with representative avian and human influenza A viruses suggests that the PB1 gene of H2N2 subtype human influenza A viruses may have been derived from a non-human, possibly avian influenza A virus by genetic reassortment. In addition, several regions of conserved amino acids with potential functional significance were identified in the deduced amino acid sequences of the polymerase proteins.

Comparison of the virologic and immunologic responses of volunteers to live avian-human influenza A H3N2 reassortant virus vaccines derived from two different avian influenza virus donors

We compared the abilities of the six internal RNA segments of two avian influenza viruses, A/Mallard/Alberta/88/76 (H3N8) and A/Mallard/NY/6750/78 (H2N2), to confer attenuation on wild-type human influenza A/Bethesda/1/85 (H3N2) virus in seronegative adult volunteers. Live avian-human influenza A reassortant virus vaccines derived from either avian virus parent were comparable in the following properties: safety, infectivity, immunogenicity, and genetic stability. Since the avian influenza A/Mallard/Alberta/76 virus offered no clear advantage as a donor virus, we will conduct our future evaluations on live influenza A virus reassortants derived from the more extensively characterized avian influenza A/Mallard/NY/78 virus.

Determination of antibody response to influenza virus surface glycoproteins by kinetic enzyme-linked immunosorbent assay.

We modified an existing enzyme-linked immunosorbent assay (ELISA) to be able to use new spectrophotometers which can measure the rate of color development in microtiter wells. This new kinetic-based ELISA (KELISA) required only a single dilution of specimen rather than the multiple dilutions required with endpoint ELISA. In addition, 10- to 100-fold-less specimen was required to perform the KELISA than the ELISA. The level of serum or nasal wash antibody against surface glycoproteins of influenza A or influenza B viruses determined by KELISA was reproducible and correlated highly with the results of endpoint ELISA or hemagglutination inhibition tests. The difference between the KELISA rates, which indicated than an antibody response to infection had occurred, was defined and was analogous to a 2.2-fold rise in titer for serum and a 3.4-fold rise in titer for nasal wash determined by endpoint ELISA. The KELISA was similar to endpoint ELISAs in its ability to detect rises in antibody level in paired serum or nasal wash specimens obtained from volunteers who received live attenuated influenza A reassortant virus vaccines. By eliminating the need for multiple dilutions, the use of KELISA offers the advantage of increasing the number of assays that can be performed by the same personnel compared with endpoint ELISA, while it maintains sensitivity and specificity.