Kutubuddin Mahmood

Correlates of Immune Protection Induced by Live, Attenuated, Cold-Adapted, Trivalent, Intranasal Influenza Virus Vaccine

The authors conducted a 2-year, multicenter, double-blind, placebo-controlled efficacy field trial of live, attenuated, cold-adapted, trivalent influenza vaccine administered by nasal spray to children 15-71 months old. Overall, vaccine was 92% efficacious at preventing culture-confirmed infection by influenza A/H3N2 and influenza B. Because influenza A/H1N1 did not cause disease during the years in which this study was conducted, the authors sought to determine vaccine efficacy and correlates of immune protection against experimental challenge with 107 TCID50 of attenuated H1N1 (vaccine strain) by intranasal spray. Prechallenge assessments included serum hemaglutination-inhibiting (HAI) antibody and nasal wash IgA antibody to H1N1. Vaccine was 83% efficacious (95% confidence interval, 60%-93%) at preventing shedding of H1N1 virus after challenge. Any serum HAI antibody or any nasal wash IgA antibody was correlated with significant protection from H1N1 infection as indicated by vaccine-virus shedding, and high efficacy against H1N1 challenge was demonstrated.

Cellular Immune Responses in Children and Adults Receiving Inactivated or Live Attenuated Influenza Vaccines

ABSTRACT The patterns of cellular immune responses induced by live attenuated influenza vaccine (LAIV) versus those of the trivalent inactivated influenza vaccine (TIV) have not been studied extensively, especially in children. The goals of this study were to evaluate the effects of TIV and LAIV immunization on cellular immunity to live influenza A virus in children and adults and to explore factors associated with variations in responses to influenza vaccines among individuals. A gamma interferon (IFN-γ) flow cytometry assay was used to measure IFN-γ-producing (IFN-γ+) NK and T cells in peripheral blood mononuclear cell cultures stimulated with a live influenza A virus strain before and after LAIV or TIV immunization of children and adults. The mean percentages of influenza A virus-specific IFN-γ+ CD4 and CD8 T cells increased significantly after LAIV, but not TIV, immunization in children aged 5 to 9 years. No increases in the mean levels of influenza A virus-reactive IFN-γ+ T cells and NK cells were observed in adults given LAIV or TIV. TIV induced a significant increase in influenza A virus-reactive T cells in 6-month- to 4-year-old children; LAIV was not evaluated in this age group. The postvaccination changes (n-fold) in the percentages of influenza A virus-reactive IFN-γ+ T and NK cells in adults were highly variable and correlated inversely with the prevaccination percentages, in particular with that of the CD56dim NK cell subset. In conclusion, our findings identify age, type of vaccine, and prevaccination levels of immune reactivity to influenza A virus as factors significantly associated with the magnitude of cellular immune responses to influenza vaccines.

Rescue of influenza B virus from eight plasmids

Influenza B virus causes a significant amount of morbidity and mortality, yet the systems to produce high yield inactivated vaccines for these viruses have lagged behind the development of those for influenza A virus. We have established a plasmid-only reverse genetics system for the generation of recombinant influenza B virus that facilitates the generation of vaccine viruses without the need for time consuming coinfection and selection procedures currently required to produce reassortants. We cloned the eight viral cDNAs of influenza B/Yamanashi/166/98, which yields relatively high titers in embryonated chicken eggs, between RNA polymerase I and RNA polymerase II transcription units. Virus was detected as early as 3 days after transfection of cocultured COS7 and Madin–Darby canine kidney cells and achieved levels of 106-107 plaque-forming units per ml of cell supernatant 6 days after transfection. The full-length sequence of the recombinant virus after passage into embryonated chicken eggs was identical to that of the input plasmids. To improve the utility of the eight-plasmid system for generating 6 + 2 reassortants from recently circulating influenza B strains, we optimized the reverse transcriptase–PCR for cloning of the hemagglutinin (HA) and neuraminidase (NA) segments. The six internal genes of B/Yamanashi/166/98 were used as the backbone to generate 6 + 2 reassortants including the HA and NA gene segments from B/Victoria/504/2000, B/Hong Kong/330/2001, and B/Hawaii/10/2001. Our results demonstrate that the eight-plasmid system can be used for the generation of high yields of influenza B virus vaccines expressing current HA and NA glycoproteins from either of the two lineages of influenza B virus.

Comparison of the Influenza Virus-Specific Effector and Memory B-Cell Responses to Immunization of Children and Adults with Live Attenuated or Inactivated Influenza Virus Vaccines

ABSTRACT Cellular immune responses to influenza virus infection and influenza virus vaccination have not been rigorously characterized. We quantified the effector and memory B-cell responses in children and adults after administration of either live attenuated (LAIV) or inactivated (TIV) influenza virus vaccines and compared these to antibody responses. Peripheral blood mononuclear cells were collected at days 0, 7 to 12, and 27 to 42 after immunization of younger children (6 months to 4 years old), older children (5 to 9 years old), and adults. Influenza virus-specific effector immunoglobulin A (IgA) and IgG circulating antibody-secreting cells (ASC) and stimulated memory B cells were detected using an enzyme-linked immunospot assay. Circulating influenza virus-specific IgG and IgA ASC were detected 7 to 12 days after TIV and after LAIV immunization. Seventy-nine percent or more of adults and older children had demonstrable IgG ASC responses, while IgA ASC responses were detected in 29 to 53% of the subjects. The IgG ASC response rate to LAIV immunization in adults was significantly higher than the response rate measured by standard serum antibody assays (26.3% and 15.8% by neutralization and hemagglutination inhibition assays, respectively). IgG ASC and serum antibody responses were relatively low in the younger children compared to older children and adults. TIV, but not LAIV, significantly increased the percentage of circulating influenza virus-specific memory B cells detected at 27 to 42 days after immunization in children and adults. In conclusion, although both influenza vaccines are effective, we found significant differences in the B-cell and antibody responses elicited after LAIV or TIV immunization in adults and older children and between young children and older age groups.

T cell-dependent production of IFN-γ by NK cells in response to influenza A virus

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-γ response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA, IFN-γ was produced by both CD56dim and CD56bright subsets of NK cells, as well as by fluA-specific T cells. Purified NK cells did not produce IFN-γ in response to fluA, while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-γ production by NK cells, which indicates that T cells are required for the IFN-γ response of NK cells. The fluA-induced IFN-γ production of NK cells was suppressed by anti–IL-2 Ab, while recombinant IL-2 replaced the helper function of T cells for IFN-γ production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell–dependent IFN-γ response of NK cells to fluA. Taken together, these results suggest that at an early stage of recurrent viral infection, NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells.

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

Abstract 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.

Multiple Gene Segments Control the Temperature Sensitivity and Attenuation Phenotypes of ca B/Ann Arbor/1/66

ABSTRACT Cold-adapted (ca) B/Ann Arbor/1/66 is the influenza B virus strain master donor virus for FluMist, a live, attenuated, influenza virus vaccine licensed in 2003 in the United States. Each FluMist vaccine strain contains six gene segments of the master donor virus; these master donor gene segments control the vaccines replication and attenuation. These gene segments also express characteristic biological traits in model systems. Unlike most virulent wild-type (wt) influenza B viruses, ca B/Ann Arbor/1/66 is temperature sensitive (ts) at 37°C and attenuated (att) in the ferret model. In order to define the minimal genetic components of these phenotypes, the amino acid sequences of the internal genes of ca B/Ann Arbor/1/66 were aligned to those of other influenza B viruses. These analyses revealed eight unique amino acids in three proteins: two in the polymerase subunit PA, two in the M1 matrix protein, and four in the nucleoprotein (NP). Using reverse genetics, these eight wt amino acids were engineered into a plasmid-derived recombinant of ca B/Ann Arbor/1/66, and these changes reverted both the ts and the att phenotypes. A detailed mutational analysis revealed that a combination of two sites in NP (A114 and H410) and one in PA (M431) controlled expression of ts, whereas these same changes plus two additional residues in M1 (Q159 and V183) controlled the att phenotype. Transferring this genetic signature to the divergent wt B/Yamanashi/166/98 strain conferred both the ts and the att phenotypes on the recombinant, demonstrating that this small, complex, genetic signature encoded the essential elements for these traits.

Analysis of the Frequencies and of the Memory T Cell Phenotypes of Human CD8+ T Cells Specific for Influenza A Viruses

We characterized the human CD8+ T cell response against influenza A viruses by a flow cytometry-based assay. Peripheral blood mononuclear cells (PBMCs) were incubated with inactivated influenza virus preparation, for 17 h, and were stained for intracellular interferon-gamma. Major histocompatibility complex class I-restricted memory CD8+ T cells specific for influenza antigens were detected in PBMCs from all 19 adult donors, at an average frequency of 0.39%. On average, 83% of influenza virus-specific CD8+ T cells expressed the differentiation-associated marker CD27, a percentage that is significantly higher than that of CD8+ T cells specific for pp65 of human cytomegalovirus (53%). These observations indicate that class I-restricted immunity against influenza A viruses is characterized by the persistence, after clearance of infection, of circulating antigen-specific CD8+ T cells. The different patterns of CD27 expression in influenza virus- and cytomegalovirus-specific CD8+ T cells suggest that influenza virus-specific memory and effector CD8+ T cells can be differentiated by phenotypic analysis.

Measuring antibody responses to a live attenuated influenza vaccine in children

Background: Hemagglutination inhibition (HAI) assay is the standard method for evaluating inactivated influenza vaccines, but no standard assay has been established for evaluating live attenuated influenza vaccines (LAIV). LAIV containing A/Beijing/262/95(H1N1) induced low serum HAI antibody responses to the antigenic variant, A/New Caledonia/20/99(H1N1) in a serologic study but provided protection against the A/New Caledonia-like viruses in a community study. Neutralization and HAI assays were compared by measuring H1N1 cross-reactive antibody responses to the LAIV in children. Methods: Sera were collected from 50 children 1–8 years of age before vaccination and 4–6 weeks after each dose of the LAIV. Antibody titers to the 3 vaccine viruses were measured by the HAI assay, whereas antibody titers against the H1N1 vaccine virus (A/Beijing/262/95) and 2 H1N1 antigenic variants (A/Shenzhen/227/95 and A/New Caledonia/20/99) were measured by the HAI and neutralization assays. Results: Initially seronegative participants were more likely to develop HAI seroconversion responses to the 3 vaccine viruses than the baseline seropositive participants (77% versus 14% for H1N1, 100% versus 20% for H3N2, 100% versus 19% for B, P < 0.01, Fishers exact test). For the H1N1 cross-reactive antibody responses, seroconversion rates measured by the neutralization assay were significantly higher than those measured by the HAI assay (95% versus 78%, P = 0.0485 for A/Beijing/262/95; 75% versus 24%, P < 0.0001 for A/Shenzhen/227/95; 51% versus 5%, P < 0.0001 for A/New Caledonia/20/99). Conclusions: The neutralization assay was more sensitive than the HAI assay for measuring H1N1 antibody responses after vaccination of children with the LAIV and may provide a better correlate of clinical protection provided by the LAIV.

Phenotypic Changes in Influenza-Specific CD8+ T Cells after Immunization of Children and Adults with Influenza Vaccines

The effect of trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV) on the phenotypes of circulating influenza-specific CD8+ T cells was analyzed by interferon (IFN)-gamma flow cytometry and tetramer staining. In adults, the expression of the T cell differentiation marker CD27 on virus-specific CD8+ T cells decreased after LAIV but increased after TIV. In children, expression of the cytotoxicity molecule perforin in influenza-specific CD8+ T cells increased after TIV but not after LAIV. Among children aged 6 months to 4 years who had not been vaccinated previously and who received 2 doses of TIV, CD27 expression decreased after each dose, whereas perforin expression increased after the second dose. These findings indicate that the phenotypic changes of influenza-specific CD8+ T cells differ depending on the type of vaccine and the age of the vaccinee. These differences are potentially affected by the different routes of vaccination and pathways of antigen presentation for TIV and LAIV.