Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated influenza virus vaccine.

Alternative strategies for vaccination against influenza that elicit both systemic antibody and mucosal IgA responses are needed to improve the efficacy in protection against infection. This study demonstrated that oral delivery of inactivated influenza vaccine with the heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli elicited the spectrum of humoral and cell-mediated responses in BALB/c mice critical for the protection and recovery from influenza virus infection. Coadministration of LT with oral influenza vaccine increased antiviral serum IgG and mucosal IgA responses compared with administration of oral influenza vaccine alone. Serum hemagglutination-inhibition and neutralizing antibodies were also augmented by LT. The adjuvant potentiated protection from infection with influenza A H3N2 viruses in mouse lower and upper respiratory tracts, enabling the use of lower doses of oral vaccine. Coadministration of LT with oral inactivated influenza vaccine induced influenza virus-specific proliferative T cells, interleukin-2 production, and major histocompatibility complex class I-restricted cytotoxic T cells.

A human chromosome 12-associated 83-kilodalton cellular protein specifically binds to the loop region of human immunodeficiency virus type 1 trans-activation response element RNA.

trans activation of human immunodeficiency virus type 1 (HIV-1) involves the viral trans-activator protein (Tat) and a cellular factor(s) encoded on human chromosome 12 (HuChr12) that targets the trans-activation response element (TAR) in the viral long terminal repeat. Because nascent TAR RNA is predicted to form a secondary structure that specifically binds cellular proteins, we investigated the composition of the TAR RNA-protein complex for HuChr12-specific proteins. UV cross-linking of TAR RNA-nuclear protein complexes formed in vitro identified an 83-kDa protein in human cells and in a human-hamster hybrid cell containing only HuChr12. The 83-kDa TAR RNA-binding protein was absent in the parental hamster cells. TAR RNA mutations that inhibited binding of the 83-kDa protein in vitro also inhibited HuChr12-dependent Tat trans activation. These TAR mutations changed the native sequence or secondary structure of the TAR loop. The TAR RNA binding activity of the 83-kDa protein also correlated with a HuChr12-dependent increase in steady-state HIV-1 RNA expression during Tat trans activation. Our results suggest that either a species-specific 83-kDa TAR RNA loop-binding protein is directly encoded on HuChr12 or a HuChr12 protein(s) induces the expression of an 83-kDa TAR-binding protein in nonprimate cells.

TAR loop-dependent human immunodeficiency virus trans activation requires factors encoded on human chromosome 12.

The trans-activator response region (TAR) RNA in the human immunodeficiency virus type 1 (HIV-1) and HIV-2 long terminal repeat forms stem-loop secondary structures in which the loop sequence is essential for trans activation. We investigated how the HIV trans-activation mechanism encoded on human chromosome 12 relates to the TAR RNA loop-dependent pathway. DNA transfection experiments showed that trans activation in human-hamster hybrid cells with the single human chromosome 12 and human T-cell lines was highly dependent on the native sequences of the HIV-1 TAR loop and the HIV-2 5' TAR loop. In nonhuman cell lines or hybrid cells without chromosome 12 that supported trans activation, the cellular mechanism was independent of the HIV-1 TAR loop and the response to mutations in the HIV-2 TAR loops differed from that found in human T-cell lines and human-hamster hybrid cells with chromosome 12. Our results suggest that the human chromosome 12 mechanism interacts directly with the TAR RNA loop or indirectly by regulating TAR RNA-binding proteins.

Human chromosome-dependent and -independent pathways for HIV-2 trans-activation.

Human immunodeficiency virus (HIV types 1 and 2) replication is controlled by the interaction of viral-encoded regulatory proteins and host cellular proteins with the viral long terminal repeat (LTR). The presence of HIV-1 and HIV-2 trans-activator proteins, tat1 and tat2, respectively, greatly increases viral gene expression from their homologous LTRs. It is unclear if the cellular factors that support tat1-directed trans-activation of the HIV-1 LTR are the same for tat2 trans-activation of the HIV-2 LTR. Human-Chinese hamster ovary hybrid cell clones were used to probe for human chromosomes involved in regulating HIV-1 and HIV-2 tat-directed transactivation. DNA transfection experiments showed that the presence of human chromosome 12 in human-hamster hybrid clones was necessary for high-level tat-directed trans-activation of the HIV-1 and -2 LTR. Cross-trans-activation of the HIV-2 LTR by tat1 was found to be chromosome 12 independent. In addition, chromosome 12 did not support trans-activation of another human retrovirus (human T-cell leukemia virus type I). Our results suggest that HIV-1 and -2 have evolved to employ a cellular pathway(s) encoded on human chromosome 12 for supporting homologous tat-directed trans-activation. Trans-activation of the HIV-2 LTR by tat1 in chromosome 12-minus cells suggests that multiple cellular pathways can be recruited to trans-activate the HIV-2 LTR and that these pathways may have been important in an HIV-like progenitor virus.

Human chromosome 12 is required for elevated HIV-1 expression in human-hamster hybrid cells.

Host cell factors act together with regulatory genes of the human immunodeficiency virus (HIV) to control virus production. Human-Chinese hamster ovary hybrid cell clones were used to probe for human chromosomes involved in regulating HIV gene expression. DNA transfection experiments showed that 4 of 18 clones had high levels of HIV gene expression measured by both extracellular virus production and transactivation of the HIV long terminal repeat in the presence of the trans-activator (tat) gene. Karyotype analyses revealed a 94% concordance (17/18) between human chromosome 12 and HIV gene expression. Other chromosomes had an 11 to 72% concordance with virus production.

Comparative studies of wild-type and cold-mutant (temperature-sensitive) influenza viruses: independent segregation of temperature-sensitivity of virus replication from temperature-sensitivity of virion transcriptase activity during recombination of mutant A/Ann Arbor/6/60 with wild-type H3N2 strains.

RNA 1 (see end of Summary) of a cold-adapted and temperature-sensitive (ts) influenza virus mutant A/Ann Arbor/6/60 has a different mobility from RNA 1 of wild-type (wt) A/Ann Arbor/6/60 when subjected to electrophoresis through acrylamide/agarose gels in the absence of denaturing agents. Detection of this lesion in RNA 1 of the mutant virus was dependent on the temperature of the gel during electrophoresis. Because RNA 1 is believed to code for a protein involved in virus-specific RNA synthesis we compared phenotypes of virion transcriptases in the wt and mutant viruses. The enzyme of the mutant virus was found to be about 40% less active at 40 degrees C than the enzyme of the wt virus when related to their activities at 31 degrees C. Two cold-adapted ts recombinants which derive their RNA 1 from the mutant A/Ann Arbor/6/60 have virion transcriptases with a phenotype similar to that of their mutant parent. Three different cold-adapted ts recombinants, however, which also derive their RNA 1 from the mutant A/Ann Arbor/6/60, have virion transcriptases with a phenotype similar to that of wt virus. We conclude, therefore, that the conditional-lethal ts property of A/Ann Arbor/6/60 mutant and its recombinants is independent of the phenotypic marker observed for the A/Ann Arbor/6/60 mutant virion transcriptase, and that the lesion in RNA 1 of the mutant may also be unrelated to the observed difference between virion transcriptases of the mutant and wt A/Ann Arbor/6/60 viruses. The phenotypes of the virion transcriptases in recombinants did, however, correlate with the derivation of their RNA 2. This suggests that the increased temperature-sensitivity of virion transcriptase of the A/Ann Arbor/6/60 mutant is caused by either (1) a lesion (not necessarily conditionally lethal) that occurred in its RNA 2 during the course of cold-adaptation, or (2) a lesion in another gene whose product is a component of the virion transcriptase complex, but which lesion is only expressed phenotypically when there is a synergistic interaction in the transcriptase complex with the product of A/Ann Arbor/6/60 rna 2.

Antigenic relationships among influenza virua A neuraminidase (N2) antigens by immunodiffusion and postinfection neutralization tests.

The antigenic relationships among the neuraminidases of influenza A strains from 1957 to 1973 were examined by postinfection application of neuraminidase antisera. This procedure causes inhibition of virus spread and apparent neutralization. Neuraminidase (apparent) neutralization and neuraminidase inhibition tests with chicken antisera gave similar results. Neuraminidase inhibition tests were more discriminating than neuraminidase neutralization tests when rabbit and goat antisera were used. Antibody absorption studies revealed that the neuraminidase, like the hemagglutinin, may possess two kinds of antigenic determinants, which can give rise to "common," or "cross-reacting," and "specific" antibodies. "Specific" antibody appears to be more effective in the inhibition of enzyme activity than in the inhibition of virus spread.

A simple double immunodiffusion test for typing influenza viruses.

The identification of influenza virus type has traditionally been based on the characterization of internal nucleoprotein (NP) antigens by the complement fixation (CF) test. Because this test is complex and time-consuming, it is used only infrequently. In this report we describe a double immunodiffusion (DID) test, which is proposed as a replacement for the CF test for the typing of influenza viruses. The DID test is simple and requires only the allantoic fluid content of a single infected embryonated egg. Virus from the infected fluid is precipitated with mild acid and disrupted by detergent. Anti-NP or anti-matrix protein (MP) antibody is equally effective in most instances for typing influenza isolates by this test. The latter is preferred, however, since it seemed to be slightly more sensitive than anti-NP antibody.