Induction of antibodies and T cell responses by a recombinant influenza virus carrying an HIV-1 TatΔ51-59 protein in mice.

Recombinant influenza viruses hold promise as vectors for vaccines to prevent transmission of mucosal pathogens. In this study, we generated a recombinant WSN/TatΔ(51-59) virus in which Tat protein lacking residues 51 to 59 of the basic domain was inserted into the N-terminus of the hemagglutinin (HA) of A/WSN/33 virus. The TatΔ(51-59) insertion into the viral HA caused a 2-log reduction in viral titers in cell culture, compared with the parental A/WSN/33 virus, and severely affected virus replication in vivo. Nevertheless, Tat-specific antibodies and T cell responses were elicited upon a single intranasal immunization of BALB/c mice with WSN/TatΔ(51-59) virus. Moreover, Tat-specific immune responses were also detected following vaccine administration via the vaginal route. These data provide further evidence that moderately large HIV antigens can be delivered by chimeric HA constructs and elicit specific immune responses, thus increasing the options for the potential use of recombinant influenza viruses, and their derivatives, for prophylactic and therapeutic vaccines.

Vaccination for seasonal influenza in patients with cancer: recommendations of the Italian Society of Medical Oncology (AIOM).

BACKGROUND: Influenza virus causes annual epidemics in the winter-spring season with significant morbidity in the general population and important mortality in high-risk groups, including cancer patients. Opinions on the suitability of patients with malignancies not undergoing active treatment and in different phases of antineoplastic therapy, to receive influenza vaccination, vary considerably among oncologists, sometimes even within one center. METHODS: We reviewed available data, including recommendations by national health authorities, on impact of influenza in patients with cancer and their capacity to mount protective immunological responses to vaccination, thus allowing, on behalf of Italian Association of Medical Oncology, to make suitable recommendations for the prevention and treatment of seasonal influenza. RESULTS AND DISCUSSION: Patients with cancer often have disease- or treatment-related immunosuppression, and as a consequence, they may have a suboptimal serologic response to influenza vaccination. The protective effect of the different preparations of influenza vaccines in patients with cancer has not been widely investigated, especially in adult patients harboring solid tumors. The optimal timing for administration of influenza vaccines in patients receiving chemotherapy is also not clearly defined. However, since vaccination is the most effective method, along with antiviral drugs in selected patients, for preventing influenza infection, it has to be recommended for cancer patients. Implementing vaccination of close contacts of oncology patients would be an additional tool for enhancing protection in fragile patient populations.

Investigation of an imported case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Florence, Italy, May to June 2013.

On 31 May 2013, the first case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Italy was laboratory confirmed in a previously healthy adult man, who developed pneumonia with moderate respiratory distress after returning from a holiday in Jordan. Two secondary cases were identified through contact tracing, among family members and colleagues who had not previously travelled abroad. Both secondary cases developed mild illness. All three patients recovered fully.

The role of antigen in the localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza pneumonia.

The role of Ag in the recruitment and localization of naive, acutely activated, and memory CD8(+) T cells to the lung during influenza infection was explored using TCR-transgenic (Tg) mice. Naive, Thy1.2(+)CD8(+) OT-I TCR-Tg cells were primed and recruited to the lung after transfer into congenic Thy1.1(+) recipients challenged with a genetically engineered influenza virus (influenza A/WSN/33 (WSN)-OVA(I)) containing the K(b) restricted OVA(257-264) epitope (siinfekl) in the viral neuraminidase stalk. However, if the transferred animals were infected with a similar influenza virus that expressed an irrelevant K(b) epitope (WSN-PEPII), no TCR-Tg T cells were detectable in the lung, although they were easily visible in the lymphoid organs. Conversely, there were substantial numbers of OT-I cells found in the lungs of WSN-PEPII-infected mice when the animals had been previously, or were concurrently, infected with a recombinant vaccinia virus expressing OVA. Similar results were obtained with nontransgenic populations of memory CD8(+) T cells reactive to a murine gamma-herpesvirus-68 Ag. Interestingly, the primary host response to the immunodominant influenza nucleoprotein epitope was not affected by the presence of memory or recently activated OT-I T cells. Thus, although Ag is required to activate the T cells, the subsequent localization of T cells to the lung during a virus infection is a property of recently activated and memory T cells and is not necessarily driven by Ag in the lung.

Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals.

Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.

Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus.

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

Postexposure vaccination massively increases the prevalence of gamma-herpesvirus-specific CD8+ T cells but confers minimal survival advantage on CD4-deficient mice.

Mice that lack CD4(+) T cells remain clinically normal for more than 60 days after respiratory challenge with the murine gamma-herpesvirus 68 (gammaHV-68), then develop symptoms of a progressive wasting disease. The gammaHV-68-specific CD8(+) T cells that persist in these I-A(b-/-) mice are unable to prevent continued, but relatively low level, virus replication. Postexposure challenge with recombinant vaccinia viruses expressing gammaHV-68 lytic cycle epitopes massively increased the magnitude of the gammaHV-68-specific CD8(+) population detectable by staining with tetrameric complexes of MHC class I glycoprotein + peptide, or by interferon-gamma production subsequent to in vitro restimulation with peptide. The boosting effect was comparable for gammaHV-68-infected I-A(b-/-) and I-A(b+/+) mice within 7 days of challenge, and took more than 110 days to return to prevaccination levels in the I-A(b+/+) controls. Although the life-span of the I-A(b-/-) mice was significantly increased, there was no effect on long-term survival. A further boost with a recombinant influenza A virus failed to improve the situation. Onset of weight loss was associated with a decline in gammaHV-68-specific CD8(+) T cell numbers, though it is not clear whether this was a cause or an effect of the underlying pathology. Even very high levels of virus-specific CD8(+) T cells thus provide only transient protection against the uniformly lethal consequences of gammaHV-68 infection under conditions of CD4(+) T cell deficiency.

A gamma-herpesvirus sneaks through a CD8(+) T cell response primed to a lytic-phase epitope.

To determine whether established CD8(+) T cell memory to an epitope prominent during the replicative phase of a gamma-herpesvirus infection protects against subsequent challenge, mice were primed with a recombinant vaccinia virus expressing the p56 peptide and then boosted by intranasal exposure to an influenza A virus incorporating p56 in the neuraminidase protein. Clonally expanded populations of functional, p56-specific CD8(+) T cells were present at high frequency in both the lung and the lymphoid tissue 1 month later, immediately before respiratory challenge with gammaHV-68. This prime-and-boost regime led to a massive reduction of productive gammaHV-68 infection in the respiratory tract and, initially, to much lower levels of latency in both the regional lymph nodes and the spleen. The CD8(+) T cell response to another epitope (p79) was diminished, there was less evidence of B cell activation, and the onset of the CD4(+) T cell-dependent splenomegaly was delayed. Within 3-4 weeks of the gammaHV-68 challenge, however, the extent of latent infection in the lymph nodes and spleen was equivalent, and both groups developed the prominent infectious mononucleosis-like syndrome that is characteristic of this infection. The reverse protocol (influenza then vaccinia) seemed to be slightly less effective. Even though immune CD8(+) T cells may be present at the time and site of virus challenge, establishing a high level of CD8(+) T cell memory to lytic-phase epitopes alone does not protect against the longer-term consequences of this gammaHV infection.

Amino acid residues contributing to the substrate specificity of the influenza A virus neuraminidase.

Influenza A viruses possess two glycoprotein spikes on the virion surface: hemagglutinin (HA), which binds to oligosaccharides containing terminal sialic acid, and neuraminidase (NA), which removes terminal sialic acid from oligosaccharides. Hence, the interplay between these receptor-binding and receptor-destroying functions assumes major importance in viral replication. In contrast to the well-characterized role of HA in host range restriction of influenza viruses, there is only limited information on the role of NA substrate specificity in viral replication among different animal species. We therefore investigated the substrate specificities of NA for linkages between N-acetyl sialic acid and galactose (NeuAcalpha2-3Gal and NeuAcalpha2-6Gal) and for different molecular species of sialic acids (N-acetyl and N-glycolyl sialic acids) in influenza A viruses isolated from human, avian, and pig hosts. Substrate specificity assays showed that all viruses had similar specificities for NeuAcalpha2-3Gal, while the activities for NeuAcalpha2-6Gal ranged from marginal, as represented by avian and early N2 human viruses, to high (although only one-third the activity for NeuAcalpha2-3Gal), as represented by swine and more recent N2 human viruses. Using site-specific mutagenesis, we identified in the earliest human virus with a detectable increase in NeuAcalpha2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the specificity for this substrate. Valine at position 275 was maintained in all later human viruses as well as swine viruses. A similar examination of N-glycolylneuraminic acid (NeuGc) specificity showed that avian viruses and most human viruses had low to moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as high as that of swine viruses. The amino acid at position 431 was found to determine the level of NeuGc specificity of NA: lysine conferred high NeuGc specificity, while proline, glutamine, and glutamic acid were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in the reaction mechanism. This finding suggests that the adaptation of NA to different substrates occurs by a mechanism of amino acid substitutions that subtly alter the conformation of NA in and around the active site to facilitate the binding of different species of sialic acid.

Influence of host species on the evolution of the nonstructural (NS) gene of influenza A viruses.

The matrix (M) and nonstructural (NS) genes of influenza A viruses each encode two overlapping proteins. In the M gene, evolution of one protein affects that of the other. To determine whether or not this evolutionary influence operating between the two M proteins also occurs in the NS gene, we sequenced the NS genes of 36 influenza A viruses isolated from a broad spectrum of animal species (wild and domestic birds, horses, pigs, humans, and sea mammals) and analyzed them phylogenetically, together with other previously published sequences. These analyses enabled us to conclude the following host species-related points that are not found in the other influenza A virus genes and their gene products. (1) The evolution of the two overlapping proteins encoded by the NS gene are lineage-dependent, unlike the M gene where evolutionary constraints on the Ml protein affect the evolution of the M2 protein (Ito et al.. J. Virol. 65 (1991) 5491 5498). (2) The gull-specific lineage contained nonH13 gull viruses and the non-gull avian lineage contained H13 gull viruses, indicating that the gull-specific lineage does not link to the H13 HA subtype in the NS gene unlike findings with other genes. (3) The branching topology of the recent equine lineage (H7N7 viruses isolated after 1973 and H3N8) indicates recent introduction of the NS, M, and PB2 genes into horses from avian sources by genetic reassortment.

Molecular basis for the generation in pigs of influenza A viruses with pandemic potential.

Genetic and biologic observations suggest that pigs may serve as "mixing vessels" for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.

The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction.

Influenza A viruses can be isolated from a variety of animals, but their range of hosts is restricted. For example, human influenza viruses do not replicate in duck intestine, the major replication site of avian viruses in ducks. Although amino acids at positions 226 and 228 of hemagglutinin (HA) of the H3 subtype are known to be important for this host range restriction, the contributions of specific amino acids at these positions to restriction were not known. Here, we address this issue by generating HAs with site-specific mutations of a human virus that contain different amino acid residues at these positions. We also let ducks select replication-competent viruses from a replication-incompetent virus containing a human virus HA by inoculating animals with 10(10.5) 50% egg infectious dose of the latter virus and identified a mutation in the HA. Our results showed that the Ser-to-Gly mutation at position 228, in addition to the Leu-to-Gln mutation at position 226 of the HA of the H3 subtype, is critical for human virus HA to support virus replication in duck intestine.

The M2 ectodomain is important for its incorporation into influenza A virions.

M2 is an integral protein of influenza A virus that functions as an ion channel. The ratio of M2 to HA in influenza A virions differs from that found on the cell surface, suggesting selective incorporation of M2 and HA into influenza virions. To examine the sequences that are important for M2 incorporation into virions, we used an incorporation assay that involves expressing M2 from a plasmid, transfecting the plasmid into recipient cells, and then infecting those cells with influenza virus. To test the importance of the different regions of the protein (extracellular, transmembrane, and cytoplasmic) in determining M2 incorporation, we created chimeric mutants of M2 and Sendai virus F proteins, exchanging corresponding extracellular, transmembrane, and cytoplasmic domains. Of the six possible chimeric mutants, only three were expressed on the cell surface. Of these three chimeric proteins, only one mutant (with the extracellular domain from M2 and the rest from F) was incorporated into influenza virions. These results suggest that the extracellular domain of M2 is important for its incorporation into virions.

The cysteine residues of the M2 protein are not required for influenza A virus replication.

The M2 protein of influenza A virus functions as an ion channel. It contains three cysteine residues: cysteines 17 and 19, which form disulfide bonds in the ectodomain, and cysteine 50 which is acylated. To understand the role of these cysteine residues in virus replication, we used reverse genetics to create influenza viruses in which the individual cysteines were mutated and a virus in which all three cysteines were changed to serine. The M2 cysteine mutants that lacked either of the cysteine residues in the ectodomain and the mutant that lacked all three residues had appreciably lower amounts of M2 oligomers than did the wild-type virus when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. None of the mutants, however, were defective in replication, either in vitro or in ferrets and mice. These findings demonstrate that noncovalent interactions are sufficient for the M2 protein to form functional oligomers for virus replication and that its cysteine residues are dispensable for influenza virus replication in vitro and in vivo.

Nuclear import and export of influenza virus nucleoprotein.

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.

HEL-Flu: an influenza virus containing the hen egg lysozyme epitope recognized by CD4+ T cells from mice transgenic for an alphabeta TCR.

Reverse genetics was used to modify the influenza virus genome by inserting the p46-63 sequence of hen egg lysozyme (HEL) into the neuraminidase stalk of the virus. The resulting virus, HEL-Flu, contained the epitopes recognized by CD4+ T cells from 3A9-TCR transgenic mice (C3HTg). Here, we show that HEL-Flu was infectious in the respiratory tract of both C3H and C3HTg mice, the latter animals showing an early, transient morbidity. Splenic dendritic cells and certain cloned populations of splenic macrophages and brain microglia constitutively presented infectious and inactivated HEL-Flu to the T cells in an Ag-specific and MHC class II-restricted manner. These results demonstrate the utility of HEL-Flu in assessing the APC activity for naive T cells; they also extend the previous studies showing that discrete populations of macrophages and microglia constitutively process and present Ag to naive T cells.

Continued evolution of H1N1 and H3N2 influenza viruses in pigs in Italy.

Swine influenza viruses possessing avian genes were first detected in Europe in 1979 (Scholtissek et al., 1983, Virology, 129, 521-523) and continue to circulate in pigs in that region of the world. To characterize the molecular epidemiology of swine influenza viruses currently circulating in Europe, we used dot-blot hybridization and sequence analysis to determine the origin of the genes encoding the nonsurface proteins ("internal" genes) of 10 H1N1 and 11 H3N2 swine influenza viruses isolated in Italy between 1992 and 1995. All of the 126 genes examined were of avian origin; thus the currently circulating H3N2 strains which possess A/Port Chalmers/1/73-like surface glycoproteins appear to be descendants of the reassortant human-avian viruses that emerged between 1983 and 1985 in Italy. Sequence analysis of matrix (M), nonstructural, and nucleoprotein genes, as well as phylogenetic analysis of M gene showed that the H1N1 and H3N2 viruses from the pigs were closely related to recent isolates of the avian-like swine H1N1 influenza strain currently circulating in northern Europe and were distinguishable from the genes of viruses isolated from European swine in 1979. To evaluate the frequency of transmission of swine H1N1 and H3N2 viruses to man, we tested 123 human sera for hemagglutination-inhibiting antibodies against avian and mammalian H1N1 and H3N2 virus strains. Our findings indicate that swine influenza viruses possessing A/Port Chalmers/1/73-like hemagglutinin may have transmitted to approximately 20% of young persons under 20 years of age who had contact with pigs. Thus, H3N2 swine viruses, possibly possessing avian-derived internal genes, may be entering humans more often than was previously thought. We strongly recommend that pigs be regularly monitored as a potential early warning system for detection of future pandemic strains.

Immunogenicity of influenza vaccine (1993-94 winter season) in HIV-seropositive and -seronegative ex-intravenous drug users.

The humoral response (haemagglutination inhibiting antibodies) to trivalent split influenza vaccine for the 1993-94 winter season (A/Beijing/32/92 (H3N2), A/Singapore/6/86 (H1N1) and B/Panama/45/90) was evaluated in a group of young HIV-seropositive ex-intravenous heroin users and compared with responses measured in HIV-seronegative individuals with a similar history. HIV-negative volunteers showed an overall positive response suggesting that previous heroin use did not influence their humoral response to influenza vaccine. Comparable results were obtained in HIV-positive subjects with CD4+ lymphocyte counts > 500 microliters-1, whereas impaired reactivity was found in HIV-positive volunteers with CD4+ counts < 500 microliters-1. Booster vaccination did not increase antibody levels in any of the groups studied, although the data did not exclude a positive influence of a second vaccine dose on persistence of antibody at 120 days after the first dose. No significant changes were observed in p24 antigenemia levels in HIV-positive individuals after vaccination.

The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication.

In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.

Reverse genetics system for generation of an influenza A virus mutant containing a deletion of the carboxyl-terminal residue of M2 protein.

We established a reverse genetics system for the M gene of influenza A virus, using amantadine resistance as a selection criterion. Transfection of an artificial M ribonucleoprotein complex of A/Puerto Rico/8/34 (H1N1), a naturally occurring amantadine-resistant virus, and superinfection with amantadine-sensitive A/equine/Miami/1/63 (H3N8), followed by cultivation in the presence of the drug, led to the generation of a transfectant virus with the A/Puerto Rico/8/34 (H1N1) M gene. With this system, we attempted to generate a virus containing a deletion in an M-gene product (M2 protein). Viruses lacking the carboxyl-terminal Glu of M2, but not those lacking 5 or 10 carboxyl-terminal residues, were rescued in the presence of amantadine. These findings indicate that carboxyl-terminal residues of the M2 protein play an important role in influenza virus replication. The M-gene-based reverse genetics system will allow the study of different M-gene mutations to achieve a balance between attenuation and virus replication, thus facilitating the production of live vaccine strains.