Variable efficacy of repeated annual influenza vaccination

Conclusions have differed in studies that have compared vaccine efficacy in groups receiving influenza vaccine for the first time to efficacy in groups vaccinated more than once. For example, the Hoskins study [Hoskins, T. W., Davis, J. R., Smith, A. J., Miller, C. L. & Allchin, A. (1979) Lancet i, 33–35] concluded that repeat vaccination was not protective in the long term, whereas the Keitel study [Keitel, W. A., Cate, T. R., Couch, R. B., Huggins, L. L. & Hess, K. R. (1997) Vaccine 15, 1114–1122] concluded that repeat vaccination provided continual protection. We propose an explanation, the antigenic distance hypothesis, and test it by analyzing seven influenza outbreaks that occurred during the Hoskins and Keitel studies. The hypothesis is that variation in repeat vaccine efficacy is due to differences in antigenic distances among vaccine strains and between the vaccine strains and the epidemic strain in each outbreak. To test the hypothesis, antigenic distances were calculated from historical hemagglutination inhibition assay tables, and a computer model of the immune response was used to predict the vaccine efficacy of individuals given different vaccinations. The model accurately predicted the observed vaccine efficacies in repeat vaccinees relative to the efficacy in first-time vaccinees (correlation 0.87). Thus, the antigenic distance hypothesis offers a parsimonious explanation of the differences between and within the Hoskins and Keitel studies. These results have implications for the selection of influenza vaccine strains, and also for vaccination strategies for other antigenically variable pathogens that might require repeated vaccination.

Chronic stress alters the immune response to influenza virus vaccine in older adults.

To determine whether a chronic stressor (caregiving for a spouse with a progressive dementia) is associated with an impaired immune response to influenza virus vaccination, we compared 32 caregivers' vaccine responses with those of 32 sex-, age-, and socioeconomically matched control subjects. Caregivers showed a poorer antibody response following vaccination relative to control subjects as assessed by two independent methods, ELISA and hemagglutination inhibition. Caregivers also had lower levels of in vitro virus-specific-induced interleukin 2 levels and interleukin 1beta; interleukin 6 did not differ between groups. These data demonstrate that down-regulation of the immune response to influenza virus vaccination is associated with a chronic stressor in the elderly. These results could have implications for vulnerability to infection among older adults.

Long term trends in the evolution of H(3) HA1 human influenza type A

We have studied the HA1 domain of 254 human influenza A(H3N2) virus genes for clues that might help identify characteristics of hemagglutinins (HAs) of circulating strains that are predictive of that strain’s epidemic potential. Our preliminary findings include the following. (i) The most parsimonious tree found requires 1,260 substitutions of which 712 are silent and 548 are replacement substitutions. (ii) The HA1 portion of the HA gene is evolving at a rate of 5.7 nucleotide substitutions/year or 5.7 × 10−3 substitutions/site per year. (iii) The replacement substitutions are distributed randomly across the three positions of the codon when allowance is made for the number of ways each codon can change the encoded amino acid. (iv) The replacement substitutions are not distributed randomly over the branches of the tree, there being 2.2 times more changes per tip branch than for non-tip branches. This result is independent of how the virus was amplified (egg grown or kidney cell grown) prior to sequencing or if sequencing was carried out directly on the original clinical specimen by PCR. (v) These excess changes on the tip branches are probably the result of a bias in the choice of strains to sequence and the detection of deleterious mutations that had not yet been removed by negative selection. (vi) There are six hypervariable codons accumulating replacement substitutions at an average rate that is 7.2 times that of the other varied codons. (vii) The number of variable codons in the trunk branches (the winners of the competitive race against the immune system) is 47 ± 5, significantly fewer than in the twigs (90 ± 7), which in turn is significantly fewer variable codons than in tip branches (175 ± 8). (viii) A minimum of one of every 12 branches has nodes at opposite ends representing viruses that reside on different continents. This is, however, no more than would be expected if one were to randomly reassign the continent of origin of the isolates. (ix) Of 99 codons with at least four mutations, 31 have ratios of non-silent to silent changes with probabilities less than 0.05 of occurring by chance, and 14 of those have probabilities <0.005. These observations strongly support positive Darwinian selection. We suggest that the small number of variable positions along the successful trunk lineage, together with knowledge of the codons that have shown positive selection, may provide clues that permit an improved prediction of which strains will cause epidemics and therefore should be used for vaccine production.

Effects of passage history and sampling bias on phylogenetic reconstruction of human influenza A evolution

In this paper we determine the extent to which host-mediated mutations and a known sampling bias affect evolutionary studies of human influenza A. Previous phylogenetic reconstruction of influenza A (H3N2) evolution using the hemagglutinin gene revealed an excess of nonsilent substitutions assigned to the terminal branches of the tree. We investigate two hypotheses to explain this observation. The first hypothesis is that the excess reflects mutations that were either not present or were at low frequency in the viral sample isolated from its human host, and that these mutations increased in frequency during passage of the virus in embryonated eggs. A set of 22 codons known to undergo such “host-mediated” mutations showed a significant excess of mutations assigned to branches attaching sequences from egg-cultured (as opposed to cell-cultured) isolates to the tree. Our second hypothesis is that the remaining excess results from sampling bias. Influenza surveillance is purposefully biased toward sequencing antigenically dissimilar strains in an effort to identify new variants that may signal the need to update the vaccine. This bias produces an excess of mutations assigned to terminal branches simply because an isolate with no close relatives is by definition attached to the tree by a relatively long branch. Simulations show that the magnitude of excess mutations we observed in the hemagglutinin tree is consistent with expectations based on our sampling protocol. Sampling bias does not affect inferences about evolution drawn from phylogenetic analyses. However, if possible, the excess caused by host-mediated mutations should be removed from studies of the evolution of influenza viruses as they replicate in their human hosts.

Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P

The polymerase (PB2) and nucleocapsid (NP) genes encoded by the genome of influenza virus are essential for replication of the virus. When synthetic genes that express RNAs for external guide sequences targeted to the mRNAs of the PB2 and NP genes are stably incorporated into mouse cells in tissue culture, infection of these cells with influenza virus is nonproductive. Endogenous RNase P cleaves the targeted influenza virus mRNAs when they are in a complex with the external guide sequences. Targeting two different mRNAs simultaneously inhibits viral particle production more efficiently than does targeting only one mRNA.

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-γ-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-γ production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-γ production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

A functionally defined model for the M2 proton channel of influenza A virus suggests a mechanism for its ion selectivity

The M2 protein from influenza A virus forms proton-selective channels that are essential to viral function and are the target of the drug amantadine. Cys scanning was used to generate a series of mutants with successive substitutions in the transmembrane segment of the protein, and the mutants were expressed in Xenopus laevis oocytes. The effect of the mutations on reversal potential, ion currents, and amantadine resistance were measured. Fourier analysis revealed a periodicity consistent with a four-stranded coiled coil or helical bundle. A three-dimensional model of this structure suggests a possible mechanism for the proton selectivity of the M2 channel of influenza virus.

Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence

Viral proteins are not naturally selected for high affinity major histocompatibility complex (MHC) binding sequences; indeed, if there is any selection, it is likely to be negative in nature. Thus, one should be able to increase viral peptide binding to MHC in the rational design of synthetic peptide vaccines. The T1 helper peptide from the HIV-1 envelope protein was made more immunogenic for inducing T cell proliferation to the native sequence by replacing a residue that exerts an adverse influence on peptide binding to an MHC class II molecule. Mice immunized with vaccine constructs combining the more potent Th helper (Th) epitope with a cytotoxic T lymphocyte (CTL) determinant developed greatly enhanced CTL responses. Use of class II MHC-congenic mice confirmed that the enhancement of CTL response was due to class II-restricted help. Thus, enhanced T cell help is key for optimal induction of CTL, and, by modification of the native immunogen to increase binding to MHC, it is possible to develop second generation vaccine constructs that enhance both Th cell activation and CTL induction.

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4°C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18°C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

Studies using double mutants of the conformational transitions in influenza hemagglutinin required for its membrane fusion activity

Amino acid substitutions widely distributed throughout the influenza hemagglutinin (HA) influence the pH of its membrane fusion activity. We have combined a number of these substitutions in double mutants and determined the effects on the pH of fusion and on the pH at which the refolding of HA required for fusion occurs. By analyzing combinations of mutations in three regions of the metastable neutral-pH HA that are rearranged at fusion pH we obtain evidence for both additive and nonadditive effects and for an apparent order of dominance in the effects of amino acid substitutions in particular regions on the pH of fusion. We conclude that there are at least three components in the structural transition required for membrane fusion activity and consider possible pathways for the transition in relation to the known differences between neutral and fusion pH HA structures.

The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer

The influenza A virus M2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells. The M2 protein is a homo-oligomer of 97 residues, and analysis by chemical cross-linking and SDS/PAGE indicates M2 forms a tetramer. However, a higher order molecular form is sometimes observed and, thus, it is necessary to determine the active form of the molecule. This was done by studying the currents of oocytes that expressed mixtures of the wild-type M2 protein (epitope tagged) and the mutant protein M2-V27S, which is resistant to the inhibitor amantadine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after antibody capture of the cell surface expressed molecules and it was found that the subunits mixed freely. When the ratio of wild-type to mutant protein subunits was 0.85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%. These results are consistent with the amantadine-resistant mutant being dominant and the oligomeric state being a tetramer.

Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia

Dissection of the primary and secondary response to an influenza A virus established that the liver contains a substantial population of CD8+ T cells specific for the immunodominant epitope formed by H-2Db and the influenza virus nucleoprotein peptide fragment NP366–374 (DbNP366). The numbers of CD8+ DbNP366+ cells in the liver reflected the magnitude of the inflammatory process in the pneumonic lung, though replication of this influenza virus is limited to the respiratory tract. Analysis of surface phenotypes indicated that the liver CD8+ DbNP366+ cells tended to be more “activated” than the set recovered from lymphoid tissue but generally less so than those from the lung. The distinguishing characteristic of the lymphocytes from the liver was that the prevalence of the CD8+ DbNP366+ set was always much higher than the percentage of CD8+ T cells that could be induced to synthesize interferon γ after short-term, in vitro stimulation with the NP366–374 peptide, whereas these values were generally comparable for virus-specific CD8+ T cells recovered from other tissue sites. Also, the numbers of apoptotic CD8+ T cells were higher in the liver. The results overall are consistent with the idea that antigen-specific CD8+ T cells are destroyed in the liver during the control and resolution phases of this viral infection, though this destruction is not necessarily an immediate process.

Influenza hemagglutinin is spring-loaded by a metastable native conformation

Enveloped viruses enter cells by protein-mediated membrane fusion. For influenza virus, membrane fusion is regulated by the conformational state of the hemagglutinin (HA) protein, which switches from a native (nonfusogenic) structure to a fusion-active (fusogenic) conformation when exposed to the acidic environment of the cellular endosome. Here we demonstrate that destabilization of HA at neutral pH, with either heat or the denaturant urea, triggers a conformational change that is biochemically indistinguishable from the change triggered by low pH. In each case, the conformational change is coincident with induction of membrane-fusion activity, providing strong evidence that the fusogenic structure is formed. These results indicate that the native structure of HA is trapped in a metastable state and that the fusogenic conformation is released by destabilization of native structure. This strategy may be shared by other enveloped viruses, including those that enter the cell at neutral pH, and could have implications for understanding the membrane-fusion step of HIV infection.

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.