Immunogenicity and cross-reactivity of a representative ancestral sequence in hepatitis C virus infection.

Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross-reactivity against widely divergent circulating strains. Rational approaches for sequence selection to maximize immunogenicity and minimize genetic distance across circulating strains may enhance vaccine induction of optimal cytotoxic T cell responses. We assessed T cell recognition of potential hepatitis C virus (HCV) vaccine sequences generated using three rational approaches: combining epitopes with predicted tight binding to the MHC, consensus sequence (most common amino acid at each position), and representative ancestral sequence that had been derived using bayesian phylogenetic tools. No correlation was seen between peptide-MHC binding affinity and frequency of recognition, as measured by an IFN-γ T cell response in HLA-matched HCV-infected individuals. Peptides encoding representative, consensus, and natural variant sequences were then tested for the capacity to expand CD8 T cell populations and to elicit cross-reactive CD8 T cell responses. CD8(+) T cells expanded with representative sequence HCV generally more broadly and robustly recognized highly diverse circulating HCV strains than did T cells expanded with either consensus sequence or naturally occurring sequence variants. These data support the use of representative sequence in HCV vaccine design.

Transcriptomes of the B and T lineages compared by multiplatform microarray profiling.

T and B lymphocytes are developmentally and functionally related cells of the immune system, representing the two major branches of adaptive immunity. Although originating from a common precursor, they play very different roles: T cells contribute to and drive cell-mediated immunity, whereas B cells secrete Abs. Because of their functional importance and well-characterized differentiation pathways, T and B lymphocytes are ideal cell types with which to understand how functional differences are encoded at the transcriptional level. Although there has been a great deal of interest in defining regulatory factors that distinguish T and B cells, a truly genomewide view of the transcriptional differences between these two cells types has not yet been taken. To obtain a more global perspective of the transcriptional differences underlying T and B cells, we exploited the statistical power of combinatorial profiling on different microarray platforms, and the breadth of the Immunological Genome Project gene expression database, to generate robust differential signatures. We find that differential expression in T and B cells is pervasive, with the majority of transcripts showing statistically significant differences. These distinguishing characteristics are acquired gradually, through all stages of B and T differentiation. In contrast, very few T versus B signature genes are uniquely expressed in these lineages, but are shared throughout immune cells.

A steady state of CD4+ T cell memory maturation and activation is established during primary subtype C HIV-1 infection.

The functional integrity of CD4(+) T cells is crucial for well-orchestrated immunity and control of HIV-1 infection, but their selective depletion during infection creates a paradox for understanding a protective response. We used multiparameter flow cytometry to measure activation, memory maturation, and multiple functions of total and Ag-specific CD4(+) T cells in 14 HIV-1- and CMV- coinfected individuals at 3 and 12 mo post HIV-1 infection. Primary HIV-1 infection was characterized by elevated levels of CD38, HLA-DR, and Ki67 in total memory and Gag-specific CD4(+) and CD8(+) T cells. In both HIV-infected and 15 uninfected controls, the frequency of activated cells was uniformly distributed among early differentiated (ED; CD45RO(+)CD27(+)), late differentiated (CD45RO(+)CD27(-)), and fully differentiated effector (CD45RO(-)CD27(-)) memory CD4(+) T cells. In HIV-1-infected individuals, activated CD4(+) T cells significantly correlated with viremia at 3 mo postinfection (r = 0.79, p = 0.0007) and also harbored more gag provirus DNA copies than nonactivated cells (p = 0.04). Moreover, Gag-specific ED CD4(+) T cells inversely associated with plasma viral load (r = -0.87, p < 0.0001). Overall, we show that low copy numbers of gag provirus and plasma RNA copies associated with low CD4 activation as well as accumulation of ED HIV-specific CD4(+) memory. Significant positive correlations between 3 and 12 mo activation and memory events highlighted that a steady state of CD4(+) T cell activation and memory maturation was established during primary infection and that these cells were unlikely to be involved in influencing the course of viremia in the first 12 mo of HIV-1 infection.

Hypertonicity-induced expression of monocyte chemoattractant protein-1 through a novel cis-acting element and MAPK signaling pathways.

MCP1 is upregulated by various stimuli, including LPS, high glucose, and hyperosmolality. However, the molecular mechanisms of transcriptional regulation of the MCP1 gene under hyperosmolar conditions are poorly understood. Treatment of NRK52E cells with NaCl or mannitol resulted in significant elevation of MCP1 mRNA and protein in a time- and dose-dependent manner. Treatment with a p38MAPK inhibitor (SB203580), an ERK inhibitor (PD98059), or an MEK inhibitor (U0126), suppressed the increase in MCP1 expression caused by hypertonic NaCl, whereas a JNK inhibitor (SP600125) and an AP1 inhibitor (curcumin) failed to attenuate MCP1 mRNA expression by NaCl. In the 5'-flanking region of the MCP1 gene, there is a sequence motif similar to the consensus TonE/ORE as well as the consensus C/E binding protein (BP), NF-kappaB, and AP1/Sp1 sites. Luciferase activity in cells transfected with reporter constructs containing a putative TonE/ORE element (MCP1-TonE/ORE) enhanced reporter gene expression under hypertonic stress. Results of electrophoretic gel mobility shift assay showed a slow migration of the MCP1-TonE/ORE probe, representing the binding of TonEBP/OREBP/NFAT5 to this enhancer element. These results indicate that the 5'-flanking region of MCP1 contains a hypertonicity-sensitive cis-acting element, MCP1-TonE/ORE, as a novel element in the MCP1 gene. Furthermore, p38MAPK and MEK-ERK pathways appear to be, at least in part, involved in hypertonic stress-mediated regulation of MCP1 expression through the MCP1-TonE/ORE.

Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA.

Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of "negatively associated" or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.

Divergent and convergent evolution after a common-source outbreak of hepatitis C virus.

The genomic sequences of viruses that are highly mutable and cause chronic infection tend to diverge over time. We report that these changes represent both immune-driven selection and, in the absence of immune pressure, reversion toward an ancestral consensus. Sequence changes in hepatitis C virus (HCV) structural and nonstructural genes were studied in a cohort of women accidentally infected with HCV in a rare common-source outbreak. We compared sequences present in serum obtained 18-22 yr after infection to sequences present in the shared inoculum and found that HCV evolved along a distinct path in each woman. Amino acid substitutions in known epitopes were directed away from consensus in persons having the HLA allele associated with that epitope (immune selection), and toward consensus in those lacking the allele (reversion). These data suggest that vaccines for genetically diverse viruses may be more effective if they represent consensus sequence, rather than a human isolate.

Cellular immune selection with hepatitis C virus persistence in humans.

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific cellular immune responses. To determine if immunologically driven sequence variation occurs with HCV persistence, we coordinately analyzed sequence evolution and CD8+ T cell responses to epitopes covering the entire HCV polyprotein in subjects who were followed prospectively from before infection to beyond the first year. There were no substitutions in T cell epitopes for a year after infection in a subject who cleared viremia. In contrast, in subjects with persistent viremia and detectable T cell responses, we observed substitutions in 69% of T cell epitopes, and every subject had a substitution in at least one epitope. In addition, amino acid substitutions occurred 13-fold more often within than outside T cell epitopes (P < 0.001, range 5-38). T lymphocyte recognition of 8 of 10 mutant peptides was markedly reduced compared with the initial sequence, indicating viral escape. Of 16 nonenvelope substitutions that occurred outside of known T cell epitopes, 8 represented conversion to consensus (P = 0.015). These findings reveal two distinct mechanisms of sequence evolution involved in HCV persistence: viral escape from CD8+ T cell responses and optimization of replicative capacity.

Fas apoptosis inhibitory molecule expression in B cells is regulated through IRF4 in a feed-forward mechanism.

Fas apoptosis inhibitory molecule (FAIM) was originally cloned as an inhibitor of Fas-mediated apoptosis in B cells that has been reported to affect multiple cell types. Recently, we found that FAIM enhances CD40L-mediated signal transduction, including induction of IFN regulatory factor (IRF)4, in vitro and augments plasma cell production in vivo. These results have keyed interest in the regulation of FAIM expression, about which little is known. Here, we show that Faim is regulated by IRF4. The Faim promoter contains three IRF binding sites, any two of which promote Faim expression. Faim promoter activity is lost following mutation of all three IRF binding sites, whereas activity of the full promoter is enhanced by concurrent expression of IRF4. In stimulated primary B cells, IRF4 expression precedes FAIM expression, IRF4 binds directly to the Faim promoter, and loss of IRF4 results in the failure of stimulated Faim up-regulation. Finally, FAIM is preferentially expressed in germinal center B cells. Taken together, these results indicate that FAIM expression is regulated through IRF4 and that this most likely occurs as part of germinal center formation. Because FAIM enhances CD40-induced IRF4 expression in B cells, these results suggest that induction of FAIM initiates a positive reinforcing (i.e., feed-forward) system in which IRF4 expression is both enhanced by FAIM and promotes FAIM expression.

Interferon (IFN) consensus sequence-binding protein, a transcription factor of the IFN regulatory factor family, regulates immune responses in vivo through control of interleukin 12 expression.

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a chronic myelogenous leukemia-like syndrome and mount impaired responses to certain viral and bacterial infections. To gain a mechanistic understanding of the contributions of ICSBP to humoral and cellular immunity, we characterized the responses of control and ICSBP-/- mice to infection with influenza A (flu) and Leishmania major (L. major). Mice of both genotypes survived infections with flu, but differed markedly in the isotype distribution of antiflu antibodies. In sera of normal mice, immunoglobulin (Ig)G2a antibodies were dominant over IgG1 antibodies, a pattern indicative of a T helper cell type 1 (Th1)-driven response. In sera of ICSBP-/- mice, however, IgG1 antibodies dominated over IgG2a antibodies, a pattern indicative of a Th2-driven response. The dominance of IgG1 and IgE over IgG2a was detected in the sera of uninfected mice as well. A seeming Th2 bias of ICSBP-deficient mice was also uncovered in their inability to control infection with L. major, where resistance is known to be dependent on IL-12 and IFN-gamma as components of a Th1 response. Infected ICSBP-deficient mice developed fulminant, disseminated leishmaniasis as a result of failure to mount a Th1-mediated curative response, although T cells remained capable of secreting IFN-gamma and macrophages of producing nitric oxide. Compromised Th1 differentiation in ICSBP-/- mice could not be attributed to hyporesponsiveness of CD4(+) T cells to interleukin (IL)-12; however, the ability of uninfected and infected ICSBP-deficient mice to produce IL-12 was markedly impaired. This indicates that ICSBP is a deciding factor in Th responses governing humoral and cellular immunity through its role in regulating IL-12 expression.

Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction.

Mice lacking the transcription factor interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family of transcription proteins, were infected with the intracellular protozoan, Toxoplasma gondii. ICSBP-deficient mice exhibited unchecked parasite replication in vivo and rapidly succumbed within 14 d after inoculation with an avirulent Toxoplasma strain. In contrast, few intracellular parasites were observed in wild-type littermates and these animals survived for at least 60 d after infection. Analysis of cytokine synthesis in vitro and in vivo revealed a major deficiency in the expression of both interferon (IFN)-gamma and interleukin (IL)-12 p40 in the T. gondii exposed ICSBP-/- animals. In related experiments, macrophages from uninfected ICSBP-/- mice were shown to display a selective impairment in the mRNA expression of IL-12 p40 but not IL-1alpha, IL-1beta, IL-1Ra, IL-6, IL-10, or TNF-alpha in response to live parasites, parasite antigen, lipopolysaccharide, or Staphylococcus aureus. This selective defect in IL-12 p40 production was observed regardless of whether the macrophages had been primed with IFN-gamma. We hypothesize that the impaired synthesis of IL-12 p40 in ICSBP-/- animals is the primary lesion responsible for the loss in resistance to T. gondii because IFN-gamma-induced parasite killing was unimpaired in vitro and, more importantly, administration of exogenous IL-12 in vivo significantly prolonged survival of the infected mice. Together these findings implicate ICSBP as a major transcription factor which directly or indirectly regulates IL-12 p40 gene activation and, as a consequence, IFN-gamma-dependent host resistance.

Regulation of apoptosis in myeloid cells by interferon consensus sequence-binding protein.

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.

IFN consensus sequence binding protein (Icsbp) is critical for eosinophil development.

IFN consensus sequence binding protein (Icsbp) (IFN response factor-8) is a hematopoietic transcription factor with dual functions in myelopoiesis and immunity. In this study, we report a novel role of Icsbp in regulating the development of eosinophils. Loss of Icsbp in mice leads to a reduction of eosinophils in different tissues. During parasite infection with the nematode Nippostrongylus brasiliensis, Icsbp-deficient mice fail to mount eosinophilia despite a vigorous IL-5 response. Numbers of phenotypically defined eosinophil progenitors are decreased and those progenitors have, on a per-cell basis, reduced eosinophil differentiation potential. The transcription factor Gata1, crucial for eosinophil development, is reduced expressed in committed eosinophil progenitors in wells as mature eosinophils. These findings identify Icsbp as a novel transcription factor critical for the development of the eosinophil lineage.

Design and characterization of a consensus hemagglutinin vaccine immunogen against H3 influenza A viruses of swine.

The substantial genetic diversity exhibited by influenza A viruses of swine (IAV-S) represents the main challenge for the development of a broadly protective vaccine against this important pathogen. The consensus vaccine immunogen has proven an effective vaccinology approach to overcome the extraordinary genetic diversity of RNA viruses. In this project, we sought to determine if a consensus IAV-S hemagglutinin (HA) immunogen would elicit broadly protective immunity in pigs. To address this question, a consensus HA gene (designated H3-CON.1) was generated from a set of 1,112 H3 sequences of IAV-S recorded in GenBank from 2011 to 2015. The consensus HA gene and a HA gene of a naturally occurring H3N2 IAV-S strain (designated H3-TX98) were expressed using the baculovirus expression system and emulsified in an oil-in-water adjuvant to be used for vaccination. Pigs vaccinated with H3-CON.1 immunogen elicited broader levels of cross-reactive neutralizing antibodies and interferon gamma secreting cells than those vaccinated with H3-TX98 immunogen. After challenge infection with a fully infectious H3N2 IAV-S isolate, the H3-CON.1-vaccinated pigs shed significantly lower levels of virus in their nasal secretions than the H3-TX98-vaccinated pigs. Collectively, our data provide a proof-of-evidence that the consensus immunogen approach may be effectively employed to develop a broadly protective vaccine against IAV-S.

Lipid transfer proteins from Rosaceae fruits share consensus epitopes responsible for their IgE-binding cross-reactivity.

Four IgE-binding epitopes have been characterized that cover a large area (40%) of the molecular surface of lipid transfer protein allergens of Rosaceae (apple, peach, apricot, and plum). They mainly correspond to electropositively charged regions protruding on the molecular surface of the modeled apple (Mal d 3), apricot (Pru ar 3), and plum (Pru d 3) allergens. Two of these epitopes consist of consensus epitopes structurally conserved among the lipid transfer protein allergens from the Rosaceae. Their occurrence in different lipid transfer protein allergens presumably accounts for the IgE-binding cross-reactivity often observed among different Rosaceae fruits. In this respect, LTP consist of phylogenetically- and structurally-related pan allergens. However, the IgE-binding cross-reactivity due to fruit lipid transfer protein has varying degrees of clinical relevance and this cross-reactivity is not necessarily accompanied by a cross-allergenicity to the corresponding fruits.

Elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence.

Dengue viruses (DENVs) are re-emerging pathogens transmitted by mosquitoes mainly in tropical and subtropical regions. Each year, they are estimated to infect 390 million people globally. The major challenge confronting dengue vaccine development is the need to induce balanced, long lasting tetravalent immune responses against four co-circulating virus serotypes (DENV-I, -II, -III, -IV), because primary infection by any one of which may predispose infected individuals to more severe diseases during a heterotypic secondary infection. Another difficulty is to select representative strains in vaccine design to provide cross-protection against most circulating virus strains. In this study, aimed at developing a tetravalent subunit vaccine with a representative single protein, we designed two vaccines (named cE80(D4) and cE80(max)) based on the consensus sequences of the ectodomain of envelope protein of 3127 DENV strains, and then expressed them in the baculovirus expression system. Both vaccines were capable of eliciting specific antibodies against all four DENV serotypes, and the predominant IgG subtype elicited by the two vaccines was IgG1. Moreover, these vaccines activated both type I and type II antigen-specific helper T cells that secreted IFN-γ and IL-4, respectively. This proof-of-concept study has set foundation for further optimization of a single protein-based tetravalent DENV vaccine.

High degree of inter-clade cross-reactivity of HIV-1-specific T cell responses at the single peptide level.

OBJECTIVES: To determine HIV-1-specific T cell responses in clade B infected individuals recognizing the clade A, B and C consensus sequences in order to assess the degree of inter-clade cross-reactivity of these immune responses at the single epitope level. METHODS: HIV-1-specific T cell responses were assessed cross-sectionally in 27 chronically HIV-1-infected individuals from a population infected mainly with clade B viral strains, using an interferon-gamma Elispot assay with a total of 1230 overlapping peptides spanning the entire amino acid sequence of the clade A, B and C 2001 consensus sequences. RESULTS: No significant difference was observed between the total magnitude or breadth of T cell responses recognizing either the clade A, B or C consensus sequences. However, at the single peptide level, 194 T cell responses were identified that recognized only one of the three different clade-specific peptide variants (A: B: C, 34: 105: 55), 125 T cell responses recognized two of the three peptide variants (AB: AC: BC, 71: 15: 39) and 166 T cell responses (34%) were cross-reactive with all three different peptide variants. Peptides recognized in all three consensus sequence variants had a significantly lower entropy (P < 0.0001) and a significantly higher inter-clade homology (P < 0.0001). CONCLUSIONS: Viral epitopes within regions of low HIV-1 clade B diversity and high inter-clade homology can be recognized in the clade A, B and C variants and indicate a wide degree of cross-isolate and cross-clade recognition by HIV-1-specific T cells. These regions may therefore be of particular relevance for the design of HIV-1 vaccines.

HIV-1 MN Env 15-mer peptides better detect HIV-1 specific CD8 T cell responses compared with consensus subtypes B and M group 15-mer peptides.

OBJECTIVE: To compare the ability of three Env (15-mer) peptide sets derived from the HIV-1 MN, the subtype B consensus, and the group M consensus to detect HIV-1 specific interferon (IFN)-gamma responses in HIV-1 subtype B infected subjects. METHODS: Peripheral blood mononuclear cells were obtained from 17 HIV-1 subtype B seropositive and 5 HIV-1 seronegative subjects. Peptide matrices comprising each peptide set were used in IFN-gamma Elispot assays to screen for T cell epitopes. Following matrix deconvolution, individual peptides were analyzed by IFN-gamma intracellular cytokine-staining to confirm and characterize the responding cells. RESULTS: HIV specific IFN-gamma responses were detected in 17 of 17 HIV-1 seropositive and none of 5 HIV-1 seronegative subjects by Elispot. Within the 17 HIV-1 seropositives, 16, 14, and 11 subjects responded to MN, B consensus, and group M env peptides, respectively. Responses were confirmed by intracellular cytokine analysis in 14 subjects and were in the CD3CD8 compartment. Cross-recognition of 'equivalent' peptides (i.e., peptides mapping to the same sequence region from the three peptide sets) was observed in 9 of 17 subjects. Peptide set specific responses to individual peptides were also observed; 11, 1, and 1 subjects demonstrated peptide set specific responses to MN, B consensus, and consensus group M, respectively. CONCLUSION: MN derived Env peptides were better able to detect HIV-1 specific CD8 T cell responses, many of which were not detectable by the equivalent clade or group consensus peptides. No single peptide set detected all the IFN-gamma responses within an individual. These results demonstrate the importance of reagent selection for monitoring of HIV responses in HIV-1 infected individuals and subsequently vaccine recipients.

Differential expression of the RTP/Drg1/Ndr1 gene product in proliferating and growth arrested cells.

Using a differential display method to identify differentiation-related genes in human myelomonocytic U937 cells, we cloned the cDNA of a gene identical to Drg1 and homologous to other recently discovered genes, respectively human RTP and Cap43 and mouse Ndr1 and TDD5 genes. Their open reading frames encode proteins highly conserved between mouse and man but which do not share homology with other know proteins. Conditions in which mRNAs are up-regulated suggest a role for the protein in cell growth arrest and terminal differentiation. We raised antibodies against a synthetic peptide reproducing a characteristic sequence of the putative polypeptide chain. These antibodies revealed a protein with the expected 43 kDa molecular mass, up-regulated by phorbol ester, retinoids and 1,25-(OH)2 vitamin D3 in U937 cells. It was increased in mammary carcinoma MCF-7 cells treated by retinoids and by the anti-estrogen ICI 182,780 but not by 4-hydroxytamoxifen. The mouse Drg1 homologous protein was up-regulated by retinoic acid in C2 myogenic cells. The diversity of situations in which expression of RTP/Drg1/Ndr1 has now been observed shows that it is widely distributed and up-regulated by various agents. Here we show that ligands of nuclear transcription factors involved in cell differentiation are among the inducers of this novel protein.

Centralized Consensus Hemagglutinin Genes Induce Protective Immunity against H1, H3 and H5 Influenza Viruses.

With the exception of the live attenuated influenza vaccine there have been no substantial changes in influenza vaccine strategies since the 1940's. Here we report an alternative vaccine approach that uses Adenovirus-vectored centralized hemagglutinin (HA) genes as vaccine antigens. Consensus H1-Con, H3-Con and H5-Con HA genes were computationally derived. Mice were immunized with Ad vaccines expressing the centralized genes individually. Groups of mice were vaccinated with 1 X 1010, 5 X 107 and 1 X 107 virus particles per mouse to represent high, intermediate and low doses, respectively. 100% of the mice that were vaccinated with the high dose vaccine were protected from heterologous lethal challenges within each subtype. In addition to 100% survival, there were no signs of weight loss and disease in 7 out of 8 groups of high dose vaccinated mice. Lower doses of vaccine showed a reduction of protection in a dose-dependent manner. However, even the lowest dose of vaccine provided significant levels of protection against the divergent influenza strains, especially considering the stringency of the challenge virus. In addition, we found that all doses of H5-Con vaccine were capable of providing complete protection against mortality when challenged with lethal doses of all 3 H5N1 influenza strains. This data demonstrates that centralized H1-Con, H3-Con and H5-Con genes can be effectively used to completely protect mice against many diverse strains of influenza. Therefore, we believe that these Ad-vectored centralized genes could be easily translated into new human vaccines.

Cloning of the non-neuronal intermediate filament protein of the gastropod Aplysia californica; identification of an amino acid residue essential for the IFA epitope.

We describe the isolation and characterization of a full-length cDNA corresponding to the larger non-neuronal (nn) intermediate filament (IF) protein of the gastropod Aplysia californica. Comparison of the sequences of the nn-IF proteins from Aplysia californica and Helix aspersa shows a strong evolutionary drift. At a 72% sequence identity level, the IF proteins of Opisthobranchia and Pulmonata show a larger distance than vimentins from Xenopus and mammals. The sequence comparison of the two snail proteins provides an important step in understanding the epitope of the monoclonal antibody IFA mapped by previous studies to the consensus sequence at the carboxy-terminal end of the rod domain of IF proteins. We identify for the first time in a naturally occurring IF protein a single amino acid exchange which leads to the loss of the epitope. The consensus sequence YRKLLEGEE present in IFA-positive proteins such as the Helix IF protein is changed in the IFA-negative Aplysia protein only by the conservative substitution of the arginine (R) by a lysine (K). Thus, the IFA epitope is not a necessity of IF structure, and its presence or absence on different IF proteins reflects only small changes in an otherwise conserved consensus sequence. Consequently, lack of IFA reactivity does not exclude the presence of IF. This result predicts that IF are much more universally expressed in lower eukaryotes than currently expected from immunological results with the monoclonal antibody IFA.