Antibody-Mediated Suppression Regulates the Humoral Immune Response to Influenza Vaccination in Humans.

BACKGROUND: Preexisting immunity, including memory B cells and preexisting antibodies, can modulate antibody responses to influenza in vivo to antigenically related antigens. We investigated whether preexisting hemagglutination inhibition (HAI) antibodies targeting the K163 epitope on the hemagglutinin (K163 antibodies) could affect antibody responses following vaccination with A/California/07/2009-like A(H1N1)pdm09 influenza viruses in humans. METHODS: Pre- and postvaccination sera collected from 300 adults (birth years, 1961-1998) in 6 seasons (2010-2016) were analyzed by HAI assays with 2 reverse genetics viruses and A(H1N1) viruses circulated from 1977 to 2018. Antibody adsorption assays were used to verify the preexisting K163 antibody-mediated suppression effect. RESULTS: Preexisting K163 antibody titers ≥80 affected HAI antibody responses following influenza vaccination containing A/California/07/2009-like antigens. At high K163 antibody concentrations (HAI antibody titers ≥160), all HAI antibody responses were suppressed. However, at moderate K163 antibody concentrations (HAI antibody titer, 80), only K163 epitope-specific antibody responses were suppressed, and novel HAI antibody responses targeting the non-K163 epitopes were induced by vaccination. Novel antibodies targeting non-K163 epitopes cross-reacted with newly emerging A(H1N1)pdm09 strains with a K163Q mutation rather than historic 1977-2007 A(H1N1) viruses. CONCLUSIONS: K163 antibody-mediated suppression shapes antibody responses to A(H1N1)pdm09 vaccination. Understanding how preexisting antibodies suppress and redirect vaccine-induced antibody responses is of great importance to improve vaccine effectiveness.

Inactivated H7 Influenza Virus Vaccines Protect Mice despite Inducing Only Low Levels of Neutralizing Antibodies.

Avian influenza viruses of the H7 hemagglutinin (HA) subtype present a significant public health threat, as evidenced by the ongoing outbreak of human A(H7N9) infections in China. When evaluated by hemagglutination inhibition (HI) and microneutralization (MN) assays, H7 viruses and vaccines are found to induce lower level of neutralizing antibodies (nAb) than do their seasonal counterparts, making it difficult to develop and evaluate prepandemic vaccines. We have previously shown that purified recombinant H7 HA appear to be poorly immunogenic in that they induce low levels of HI and MN antibodies. In this study, we immunized mice with whole inactivated reverse genetics reassortant (RG) viruses expressing HA and neuraminidase (NA) from 3 different H7 viruses [A/Shanghai/2/2013(H7N9), A/Netherlands/219/2003(H7N7), and A/New York/107/2003(H7N2)] or with human A(H1N1)pdm09 (A/California/07/2009-like) or A(H3N2) (A/Perth16/2009) viruses. Mice produced equivalent titers of antibodies to all viruses as measured by enzyme-linked immunosorbent assay (ELISA). However, the antibody titers induced by H7 viruses were significantly lower when measured by HI and MN assays. Despite inducing very low levels of nAb, H7 vaccines conferred complete protection against homologous virus challenge in mice, and the serum antibodies directed against the HA head region were capable of mediating protection. The apparently low immunogenicity associated with H7 viruses and vaccines may be at least partly related to measuring antibody titers with the traditional HI and MN assays, which may not provide a true measure of protective immunity associated with H7 immunization. This study underscores the need for development of additional correlates of protection for prepandemic vaccines.IMPORTANCE H7 avian influenza viruses present a serious risk to human health. Preparedness efforts include development of prepandemic vaccines. For seasonal influenza viruses, protection is correlated with antibody titers measured by hemagglutination inhibition (HI) and virus microneutralization (MN) assays. Since H7 vaccines typically induce low titers in HI and MN assays, they have been considered to be poorly immunogenic. We show that in mice H7 whole inactivated virus vaccines (WIVs) were as immunogenic as seasonal WIVs, as they induced similar levels of overall serum antibodies. However, a larger fraction of the antibodies induced by H7 WIV was nonneutralizing in vitro Nevertheless, the H7 WIV completely protected mice against homologous viral challenge, and antibodies directed against the HA head were the major contributor toward immune protection. Vaccines against H7 avian influenza viruses may be more effective than HI and virus neutralization assays suggest, and such vaccines may need other methods for evaluation.

Evolution and Virulence of Influenza A Virus Protein PB1-F2.

PB1-F2 is an accessory protein of most human, avian, swine, equine, and canine influenza A viruses (IAVs). Although it is dispensable for virus replication and growth, it plays significant roles in pathogenesis by interfering with the host innate immune response, inducing death in immune and epithelial cells, altering inflammatory responses, and promoting secondary bacterial pneumonia. The effects of PB1-F2 differ between virus strains and host species. This can at least partially be explained by the presence of multiple PB1-F2 sequence variants, including premature stop codons that lead to the expression of truncated PB1-F2 proteins of different lengths and specific virulence-associated residues that enhance susceptibility to bacterial superinfection. Although there has been a tendency for human seasonal IAV to gradually reduce the number of virulence-associated residues, zoonotic IAVs contain a reservoir of PB1-F2 proteins with full length, virulence-associated sequences. Here, we review the molecular mechanisms by which PB1-F2 may affect influenza virulence, and factors associated with the evolution and selection of this protein.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice.

UNLABELLED: Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE: Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

RIG-I ligand enhances the immunogenicity of recombinant H7HA protein.

Avian H7N9 influenza virus infection with fatal outcomes continues to pose a pandemic threat and highly immunogenic vaccines are urgently needed. In this report we show that baculovirus-derived recombinant H7 hemagglutinin protein, when delivered with RIG-I ligand, induced enhanced antibody and T cell responses and conferred protection against lethal challenge with a homologous H7N9 virus. These findings indicate the potential utility of RIG-I ligands as vaccine adjuvants to increase the immunogenicity of recombinant H7 hemagglutinin.

Molecular determinants of influenza virus pathogenesis in mice.

Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account.

A distinct lineage of influenza A virus from bats.

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

Cutting Edge: Coding single nucleotide polymorphisms of endoplasmic reticulum aminopeptidase 1 can affect antigenic peptide generation in vitro by influencing basic enzymatic properties of the enzyme.

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis-Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis-Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant K(i), further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

Effects of Extended-Release Buprenorphine on Mouse Models of Influenza.

Mice are widely used as small animal models for influenza infection and immunization studies because of their susceptibility to many strains of influenza, obvious clinical signs of infection, and ease of handling. Analgesia is rarely used in such studies even if nonstudy effects such as fight wounds, tail injuries, or severe dermatitis would otherwise justify it because of concerns that treatment might have confounding effects on primary study parameters such as the course of infection and/or the serological response to infection. However, analgesia for study-related or -unrelated effects may be desirable for animal welfare purposes. Opioids, such as extended-release buprenorphine, are well-characterized analgesics in mice and may have fewer immune-modulatory effects than other drug classes. In this study, BALB/c and DBA/2 mice were inoculated with influenza virus, and treatment groups received either no analgesics or 2 doses of extended-release buprenorphine 72 h apart. Clinical signs, mortality, and influenza-specific antibody responses were comparable in mice that did or did not receive buprenorphine. We therefore conclude that extended-release buprenorphine can be used to alleviate incidental pain during studies of influenza infection without altering the course of infection or the immune response.

Use of Biolayer Interferometry to Identify Dominant Binding Epitopes of Influenza Hemagglutinin Protein of A(H1N1)pdm09 in the Antibody Response to 2010-2011 Influenza Seasonal Vaccine.

The globular head domain of influenza virus surface protein hemagglutinin (HA1) is the major target of neutralizing antibodies elicited by vaccines. As little as one amino acid substitution in the HA1 can result in an antigenic drift of influenza viruses, indicating the dominance of some epitopes in the binding of HA to polyclonal serum antibodies. Therefore, identifying dominant binding epitopes of HA is critical for selecting seasonal influenza vaccine viruses. In this study, we have developed a biolayer interferometry (BLI)-based assay to determine dominant binding epitopes of the HA1 in antibody response to influenza vaccines using a panel of recombinant HA1 proteins of A(H1N1)pdm09 virus with each carrying a single amino acid substitution. Sera from individuals vaccinated with the 2010-2011 influenza trivalent vaccines were analyzed for their binding to the HA1 panel and hemagglutination inhibition (HI) activity against influenza viruses with cognate mutations. Results revealed an over 50% reduction in the BLI binding of several mutated HA1 compared to the wild type and a strong correlation between dominant residues identified by the BLI and HI assays. Our study demonstrates a method to systemically analyze antibody immunodominance in the humoral response to influenza vaccines.

Influenza A Virus Multicycle Replication Yields Comparable Viral Population Emergence in Human Respiratory and Ocular Cell Types.

While primarily considered a respiratory pathogen, influenza A virus (IAV) is nonetheless capable of spreading to, and replicating in, numerous extrapulmonary tissues in humans. However, within-host assessments of genetic diversity during multicycle replication have been largely limited to respiratory tract tissues and specimens. As selective pressures can vary greatly between anatomical sites, there is a need to examine how measures of viral diversity may vary between influenza viruses exhibiting different tropisms in humans, as well as following influenza virus infection of cells derived from different organ systems. Here, we employed human primary tissue constructs emulative of the human airway or corneal surface, and we infected both with a panel of human- and avian-origin IAV, inclusive of H1 and H3 subtype human viruses and highly pathogenic H5 and H7 subtype viruses, which are associated with both respiratory disease and conjunctivitis following human infection. While both cell types supported productive replication of all viruses, airway-derived tissue constructs elicited greater induction of genes associated with antiviral responses than did corneal-derived constructs. We used next-generation sequencing to examine viral mutations and population diversity, utilizing several metrics. With few exceptions, generally comparable measures of viral diversity and mutational frequency were detected following homologous virus infection of both respiratory-origin and ocular-origin tissue constructs. Expansion of within-host assessments of genetic diversity to include IAV with atypical clinical presentations in humans or in extrapulmonary cell types can provide greater insight into understanding those features most prone to modulation in the context of viral tropism. IMPORTANCE Influenza A virus (IAV) can infect tissues both within and beyond the respiratory tract, leading to extrapulmonary complications, such as conjunctivitis or gastrointestinal disease. Selective pressures governing virus replication and induction of host responses can vary based on the anatomical site of infection, yet studies examining within-host assessments of genetic diversity are typically only conducted in cells derived from the respiratory tract. We examined the contribution of influenza virus tropism on these properties two different ways: by using IAV associated with different tropisms in humans, and by infecting human cell types from two different organ systems susceptible to IAV infection. Despite the diversity of cell types and viruses employed, we observed generally similar measures of viral diversity postinfection across all conditions tested; these findings nonetheless contribute to a greater understanding of the role tissue type contributes to the dynamics of virus evolution within a human host.

The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins.

The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3' end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-ß) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.

Characterizing the specificity and cooperation of aminopeptidases in the cytosol and endoplasmic reticulum during MHC class I antigen presentation.

Many MHC class I-binding peptides are generated as N-extended precursors during protein degradation by the proteasome. These peptides can subsequently be trimmed by aminopeptidases in the cytosol and/or the endoplasmic reticulum (ER) to produce mature epitope. However, the contribution and specificity of each of these subcellular compartments in removing N-terminal amino acids for Ag presentation is not well defined. In this study, we investigated this issue for antigenic precursors that are expressed in the cytosol. By systematically varying the N-terminal flanking sequences of peptides, we show that the amino acids upstream of an epitope precursor are a major determinant of the amount of Ag presentation. In many cases, MHC class I-binding peptides are produced through sequential trimming in the cytosol and ER. Trimming of flanking residues in the cytosol contributes most to sequences that are poorly trimmed in the ER. Because N-terminal trimming has different specificity in the cytosol and ER, the cleavage of peptides in both of these compartments serves to broaden the repertoire of sequences that are presented.

Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1.

All three members of the oxytocinase subfamily of M1 aminopeptidases, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and placental leucine aminopeptidase (PLAP), also known as insulin-regulated aminopeptidase, have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation, whereas PLAP has been recently implicated in cross-presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in Ag processing. ERAP1 can trim a large variety of long peptide sequences and efficiently accumulate mature antigenic epitopes of 8-9 aa long. In this study, we evaluate the ability of PLAP to process antigenic peptide precursors in vitro and compare it with ERAP1. We find that, similar to ERAP1, PLAP can trim a variety of long peptide sequences efficiently and, in most cases, accumulates appreciable amounts of correct length mature antigenic epitope. Again, similar to ERAP1, PLAP continued trimming some of the epitopes tested and accumulated smaller products effectively destroying the epitope. However, the intermediate accumulation properties of ERAP1 and PLAP are distinct and epitope dependent, suggesting that these two enzymes may impose different selective pressures on epitope generation. Overall, although PLAP has the necessary enzymatic properties to participate in generating or destroying MHC class I-presented peptides, its trimming behavior is distinct from that of ERAP1, something that supports a separate role for these two enzymes in Ag processing.

Aberrant Cellular Glycosylation May Increase the Ability of Influenza Viruses to Escape Host Immune Responses through Modification of the Viral Glycome.

Individuals with metabolic dysregulation of cellular glycosylation often experience severe influenza disease, with a poor immune response to the virus and low vaccine efficacy. Here, we investigate the consequences of aberrant cellular glycosylation for the glycome and the biology of influenza virus. We transiently induced aberrant N-linked glycosylation in cultured cells with an oligosaccharyltransferase inhibitor, NGI-1. Cells treated with NGI-1 produced morphologically unaltered viable influenza virus with sequence-neutral glycosylation changes (primarily reduced site occupancy) in the hemagglutinin and neuraminidase proteins. Hemagglutinin with reduced glycan occupancy required a higher concentration of surfactant protein D (an important innate immunity respiratory tract collectin) for inhibition compared to that with normal glycan occupancy. Immunization of mice with NGI-1-treated virus significantly reduced antihemagglutinin and antineuraminidase titers of total serum antibody and reduced hemagglutinin protective antibody responses. Our data suggest that aberrant cellular glycosylation may increase the risk of severe influenza as a result of the increased ability of glycome-modified influenza viruses to evade the immune response. IMPORTANCE People with disorders such as cancer, autoimmune disease, diabetes, or obesity often have metabolic dysregulation of cellular glycosylation and also have more severe influenza disease, a reduced immune response to the virus, and reduced vaccine efficacy. Since influenza viruses that infect such people do not show consistent genomic variations, it is generally assumed that the altered biology is mainly related to host factors. However, since host cells are responsible for glycosylation of influenza virus hemagglutinin and neuraminidase, and glycosylation is important for interactions of these proteins with the immune system, the viruses may have functional differences that are not reflected by their genomic sequence. Here, we show that imbalanced cellular glycosylation can modify the viral glycome without genomic changes, leading to reduced innate and adaptive host immune responses to infection. Our findings link metabolic dysregulation of host glycosylation to increased risk of severe influenza and reduced influenza virus vaccine efficacy.

Low quality antibody responses in critically ill patients hospitalized with pandemic influenza A(H1N1)pdm09 virus infection.

Although some adults infected with influenza 2009 A(H1N1)pdm09 viruses mounted high hemagglutination inhibition (HAI) antibody response, they still suffered from severe disease, or even death. Here, we analyzed antibody profiles in patients (n = 31, 17-65 years) admitted to intensive care units (ICUs) with lung failure and invasive mechanical ventilation use due to infection with A(H1N1)pdm09 viruses during 2009-2011. We performed a comprehensive analysis of the quality and quantity of antibody responses using HAI, virus neutralization, biolayer interferometry, enzyme-linked-lectin and enzyme-linked immunosorbent assays. At time of the ICU admission, 45% (14/31) of the patients had HAI antibody titers ≥ 80 in the first serum (S1), most (13/14) exhibited narrowly-focused HAI and/or anti-HA-head binding antibodies targeting single epitopes in or around the receptor binding site. In contrast, 42% (13/31) of the patients with HAI titers ≤ 10 in S1 had non-neutralizing anti-HA-stem antibodies against A(H1N1)pdm09 viruses. Only 19% (6/31) of the patients showed HA-specific IgG1-dominant antibody responses. Three of 5 fatal patients possessed highly focused cross-type HAI antibodies targeting the (K130 + Q223)-epitopes with extremely low avidity. Our findings suggest that narrowly-focused low-quality antibody responses targeting specific HA-epitopes may have contributed to severe infection of the lower respiratory tract.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 µg hemagglutinin (HA)/dose + AS03A; 2. inactivated vaccine with 5.75 µg HA/dose + AS03A; 3. inactivated vaccine with 11.5 µg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 µg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Identification of a critical T(Q/D/E)x5ADx2(I/L) motif from primate lentivirus Vif proteins that regulate APOBEC3G and APOBEC3F neutralizing activity.

Primate lentiviruses are unique in that they produce several accessory proteins to help in the establishment of productive viral infection. The major function of these proteins is to clear host resistance factors that inhibit viral replication. Vif is one of these proteins. It functions as an adaptor that binds to the cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) and bridges them to a cullin 5 (Cul5) and elongin (Elo) B/C E3 ubiquitin ligase complex for proteasomal degradation. So far, 11 discontinuous domains in Vif have been identified that regulate this degradation process. Here we report another domain, T(Q/D/E)x(5)ADx(2)(I/L), which is located at residues 96 to 107 in the human immunodeficiency virus type 1 (HIV-1) Vif protein. This domain is conserved not only in all HIV-1 subtypes but also in other primate lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV), which infects rhesus macaques (SIVmac) and African green monkeys (SIVagm). Mutations of the critical residues in this motif seriously disrupted Vif's neutralizing activity toward both A3G and A3F. This motif regulates Vif interaction not only with A3G and A3F but also with Cul5. When this motif was inactivated in the HIV-1 genome, Vif failed to exclude A3G and A3F from virions, resulting in abortive HIV replication in nonpermissive human T cells. Thus, T(Q/D/E)x(5)ADx(2)(I/L) is a critical functional motif that directly supports the adaptor function of Vif and is an attractive target for inhibition of Vif function.

Identification of 81LGxGxxIxW89 and 171EDRW174 domains from human immunodeficiency virus type 1 Vif that regulate APOBEC3G and APOBEC3F neutralizing activity.

The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, (81)LGxGxSIEW(89) and (171)EDRWN(175). Within the (81)LGxGxSIEW(89) sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues (86)SIEW(89) affect Vif binding to A3F, A3G, and Cul5. Accordingly, this (81)LGxGxSIEW(89) sequence was designated the (81)LGxGxxIxW(89) domain. Within the (171)EDRWN(175) sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated (171)EDRW(174). The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, (81)LGxGxxIxW(89) and (171)EDRW(174) are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.

The specificity of trimming of MHC class I-presented peptides in the endoplasmic reticulum.

Aminopeptidases in the endoplasmic reticulum (ER) can cleave antigenic peptides and in so doing either create or destroy MHC class I-presented epitopes. However, the specificity of this trimming process overall and of the major ER aminopeptidase ERAP1 in particular is not well understood. This issue is important because peptide trimming influences the magnitude and specificity of CD8 T cell responses. By systematically varying the N-terminal flanking sequences of peptides in a cell-free biochemical system and in intact cells, we elucidated the specificity of ERAP1 and of ER trimming overall. ERAP1 can cleave after many amino acids on the N terminus of epitope precursors but does so at markedly different rates. The specificity seen with purified ERAP1 is similar to that observed for trimming and presentation of epitopes in the ER of intact cells. We define N-terminal sequences that are favorable or unfavorable for Ag presentation in ways that are independent from the epitopes core sequence. When databases of known presented peptides were analyzed, the residues that were preferred for the trimming of model peptide precursors were found to be overrepresented in N-terminal flanking sequences of epitopes generally. These data define key determinants in the specificity of Ag processing.