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Biomedical subjects

J I Mullins

Publications and source records attributed to J I Mullins.

At least 19 recordsLinked to original sources
PubMed1997

Mother-to-infant transmission of human immunodeficiency virus type 1 involving five envelope sequence subtypes.

Genetic analysis of human immunodeficiency virus type 1 (HIV-1) from cases of mother-to-infant transmission were analyzed in an effort to provide insights into the viral selection that may occur during transmission, as well as the timing and source of transmitted viruses. HIV-1 env genes obtained from seven mothers and their perinatally infected infants in Sweden were studied. Five envelope sequence clades (A to E) were found to be represented. We used a heteroduplex tracking assay (HTA) to assess the genetic relatedness between early viral isolates from the infants and serial maternal virus populations taken during pregnancy and at delivery. HTA findings were used to select for DNA sequence analysis maternal virus populations that were either closely or more distantly related to the infant virus. In each case, nucleotide sequence analysis confirmed the genetic relationships inferred by the HTA. Only maternal peripheral blood was sampled, and large sets of maternal specimens throughout pregnancy were generally not available. However, no consistent correlation was found to support the hypothesis that infant viruses should match blood-derived maternal virus genotypes found early in pregnancy if infants were found to be infected at birth or, conversely, that infant viruses should match blood-derived maternal virus genotypes found at delivery if infants were found to be infected only some time later.

Amino Acid Sequence↗
PubMed1997

Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and distinct evolutionary rates among envelope sequence clades.

For the rapid genetic analysis of feline immunodeficiency virus (FIV), we developed a heteroduplex mobility assay (HMA) that utilizes a PCR-amplified fragment of the FIV envelope gene spanning the third and fourth variable regions of the envelope surface protein coding sequence. Viral sequences were successfully amplified from blood specimens from 98 naturally infected cats from Australia, Canada, Germany, Italy, South Africa, and the United States. Eighty were clearly assignable to the A or B envelope sequence subtypes. Three belonged to subtype C, one was dually infected with viruses harboring the A and B env subtypes, and several were categorized as outliers to any of the established subtypes or as probable intersubtype recombinants. Some geographic clustering was evident, with subtypes A and B found in greater frequency in the western and eastern regions of the United States, respectively. Subtypes A, B, and C were found on more than one continent, and countries with more than two samples analyzed contained at least two subtypes. The broadest representation of subtypes was found in Munich, Germany, where three subtypes and one virus that was not classifiable by HMA were found. Thirteen samples were selected for DNA sequence determination over the same region of env used for HMA. Analysis of all available FIV env sequences from this and previous studies revealed the existence of recombinant viruses generated from subtype A/B, B/D, and A/C envelope gene sequences. Subtype A env sequences were less diverse than subtype B sequences, although both groups had well-supported clusters. Furthermore, the mutational pattern giving rise to diversification in the two subtypes differed, with the subtype A viruses showing half as many synonymous site mutations compared to subtype B yet showing similar levels of nonsynonymous site changes. These results are consistent with the hypothesis that FIV-B is an older virus group and is possibly more host adapted than FIV-A.

Amino Acid Sequence↗
PubMed1997

Divergent patterns of progression to AIDS after infection from the same source: human immunodeficiency virus type 1 evolution and antiviral responses.

The rate of progression to AIDS in human immunodeficiency virus type 1 (HIV-1)-infected individuals is determined by a complex series of interactions between the host and virus. Here we evaluate virologic properties and host responses in two men near-simultaneously infected with HIV-1 from the same sexual partner--one individual progressed to AIDS in less than 2 years, and the other remains asymptomatic 3 years postinfection. Distinct neutralizing antibody and cellular immune responses were evident, with the slower progressor exhibiting generally stronger and broader responses, except for cytotoxic T-lymphocyte responses early in infection. Virtually identical, homogeneous virus populations were found in both patients in the first sample obtained; however, a second unrelated HIV-1 virus population was also found in the fast progressor. Whether the second population emanated from an additional source of infection or the two were transmitted from the original source could not be determined. The virus population in the slower progressor turned over and diversified rapidly, whereas both virus populations in the rapid progressor evolved at a much slower rate. In addition, the character of mutational changes underlying these diversities appeared to be distinct, with a bias for diversifying selection developing in the slower progressor and a reciprocal bias towards purifying selection maintained in both populations in the fast progressor. Thus, the rapid evolution that is a hallmark of HIV replication may be a reflection of strong host resistance against emerging virus variants and a longer period of asymptomatic infection. Furthermore, rapid progression was not linked to a collapse of any appreciable immune response following attainment of some threshold of antigenic diversity but rather to a failure to drive this diversification and a condition of relatively unimpeded expansion of variants with optimized replicative capacity within the host.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Human immunodeficiency virus type 1 molecular evolution and the measure of selection.

Human immunodeficiency virus (HIV) envelope genes are highly variable between and often within individuals. Part of this variability is thought to be the result of immune-mediated positive selection for sequence diversity. To measure positive selection it has become customary in HIV research to calculate the ratio of the proportions of synonymous (ds) and nonsynonymous (dn) substitutions per potential synonymous or nonsynonymous site, respectively. However, another measure that can be used is the difference between ds and dn, delta d. We show, by example, that using the ratio, ds/dn, or the difference, delta d, may lead us to different conclusions regarding the existence of positive selection pressure. We conclude by noting that until we understand the processes that mediate nucleotide variation in a host selective environment, inferences based on summary statistics characterizing types of nucleotide substitutions should be made with caution.

Base Sequence↗
PubMed1996

Covariability of V3 loop amino acids.

We reanalyzed for covariability a set of 308 human immunodeficiency virus type 1 (HIV-1) V3 loop amino acid sequences from the B envelope sequence subtype previously analyzed by Korber et al.,1 as well as a new set of 440 sequences that also included substantial numbers of sequences from subtypes A, D, and E. We used the measure employed by Korber et al., essentially the likelihood ratio statistic for independence, plus two additional measures as well as clade information to examine the new set and both data sets simultaneously. We set forth the following conclusions and observations. The eight most highly connected sites identified through these statistical approaches included all of the six residues previously shown to have determining roles in structure, immunologic recognition, virus phenotype, and host range; each of the seven pairs of covariant sites found by Korber were signaled by our additional two measures in the set of 308 sequences, although 2 or 3 dropped out of the examination of the set of 440 when the requirement of stringent significance was applied for some or all of the three tests, respectively; using the same criteria, a total of 20 (including 5 Korber et al. pairs) or a total of 6 (including 4 Korber et al. pairs) were found when the set of 440 was added. Several limitations to statistical analysis of this type of HIV sequence data were also noted. For example, the data sets were, by historical necessity, collected haphazardly. For example, it was not possible to separate substantially sized groups out according to time of or since infection, disease status, antiviral treatment, geography, etc. There was also an enormous "wealth of significance" within the data. For example, for one measure the 440 data set showed 233 of the 465 pairs of sites with a likelihood ratio statistic of < 0.001. Last, most sites had consensus amino acids in 80% or more of the sequences; hence, there was an absence of data on many combinations of amino acids. Given the observed linkage between sites shown to be covariable and those known to have critical biological function, the statistical approaches we and Korber et al. have outlined may find use in predicting critical structural features of HIV proteins as targets for therapeutic intervention.

Algorithms↗
PubMed1996

Analysis of FeLV-FAIDS provirus burden and productive infection in lymphocyte subsets in vivo.

To help elucidate the immunopathogenesis of feline leukemia virus (FeLV)-induced immunodeficiency we studied the tropism of viruses derived from the FeLV-FAIDS isolate for lymphocyte subpopulations in cats. FeLV-FAIDS is composed of a replication-competent virus typical of subgroup A FeLV (prototype, clone 61E) and a family of replication-defective but immunopathogenic variant viruses (prototype, clone 61C). We sorted CD4+, CD8+, and IgG+ lymphocytes to > or = 97% purity and analyzed viral load in each cell population via genome-specific semiquantitative PCR. Both the 61E and 61C viruses were tropic for CD4+ and CD8+ T cells as well as IgG+ B lymphocytes in blood and lymph node. High provirus burden were established for both virus genomes-ranging from 0.3 to > 2 copies/cell. To identify the fraction of circulating cells which expressed viral antigen in vivo, we developed a flow cytometric method to simultaneously label blood leukocytes for surface immunophenotype and intracytoplasmic FeLV CA (p27 Gag). These experiments established that 20 to 60% of CD4+, CD8+, and IgG+ lymphocytes and > 85% of monocytes and granulocytes expressed FeLV p27 intracellularly. Thus the in vivo target cells for FeLV-FAIDS infection are manifold and include CD4+ and CD8+ T cells, B cells, and myeloid cells.

Animals↗
PubMed1996

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

AIDS Vaccines↗
PubMed1996

Efficacy of an inactivated feline leukemia virus vaccine.

An inactivated whole-virus FeLV vaccine, developed from a molecularly cloned FeLV isolate (FeLV-61E-A), was assessed for its ability to protect cats against homologous and heterologous virulent viral challenge. The fractions of cats that resisted the induction of persistent viremia after FeLV challenge were as follows: FeLV-61E-A vaccine, 95%; adjuvant controls, 26%; and established commercial control FeLV vaccine, 35%. The prechallenge mean neutralizing antibody titers for each group were as follows: FeLV-61E-A vaccine, 1:43; adjuvant controls, < 1:8; and commercial control FeLV vaccine, 1:12. The prototype FeLV-61E-A vaccine was developed commercially for immunization of pet cats by substitution of a proprietary adjuvant and development of stable, high antigen production cell lines. This vaccine (Fel-O-Vax) has been studied extensively, alone and in multivalent combination with other feline virus vaccines, in seven efficacy trials involving a total of 150 immunized cats. These studies yielded an FeLV-resistant fraction of 87% in vaccinated cats as compared with 8% in adjuvant controls. The duration of immunity induced by an FeLV-61E-A commercial vaccine (Fel-O-Vax-LvK IV) was also assessed. One year after vaccination, 100% of challenged vaccinated cats and none of challenged controls resisted induction of persistent viremia. The results of these studies demonstrate that an inactivated FeLV vaccine prepared from a molecularly cloned subgroup A FeLV produces a high level of protective immunity against heterologous and homologous FeLV infection. This vaccine-induced immunity is durable for at least 1 year without intervening booster immunization or exposure to virus.

Animals↗
PubMed1996

Human immunodeficiency virus in plasma and genital secretions during the menstrual cycle.

Six human immunodeficiency virus (HIV)-positive women were studied weekly over 8 weeks to detect HIV RNA in plasma and cervical secretions and proviral DNA in cervical, vaginal, and cervicovaginal lavage samples by polymerase chain reaction (PCR) amplification techniques. In cervical swab samples, cell-free HIV RNA was detected more frequently than cell-associated HIV proviral DNA (22/48 vs. 7/48, respectively). Cervical HIV RNA was consistently detected in 2 women with plasma HIV RNA > 100,000 copies/mL but was not detected in 2 women with plasma HIV RNA < 10,000 copies/mL, regardless of menstruation status. HIV-specific IgA was detected in the plasma of 2 women and in at least 1 cervicovaginal lavage sample from all 6 women. Thus, quantitation of cervical HIV RNA can be accomplished by PCR techniques and may be useful in evaluation genital viral shedding.

Adult↗
PubMed1996

Distribution of HIV-1 subtypes seen in an AIDS clinic in Sao Paulo City, Brazil.

OBJECTIVE: To determine the distribution of HIV-1 subtypes in Sao Paulo, Brazil. METHODS: Samples were obtained from 80 consecutive HIV-1-infected individuals attending the Immunodeficiency Clinic at the University of Sao Paulo in 1993. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque gradient and a portion was used for routine CD4 counts; the remainder were frozen. PBMC were proteinase-K-digested and DNA-purified by organic extraction. Samples were amplified for the env region of HIV, and envelope sequence subtypes determined by heteroduplex mobility analysis using prototypic subtypes as references. A subset of these were also sequenced through the C2-V3 region of env. RESULTS: A total 69 of 80 samples yielded env polymerase chain reaction product enabling subtype determination; samples that did not amplify were those with low DNA yields. Among 12 injecting drug users (IDU) or sexual partners of IDU, four were typed as clade F and eight as clade B. Forty-three homosexual men or female sexual partners of bisexual men were typed as clade B. The 14 additional cases without known risk factors were typed as clade B. CONCLUSION: These data suggest that subtype F is related to injecting drug use in Brazil.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Replication kinetics and cell tropism of an immunosuppressive feline leukaemia virus.

To elucidate in vivo cell tropism and infection kinetics of an immunodeficiency-inducing isolate of feline leukaemia virus (FeLV-FAIDS), we quantified the two major genotypes comprising FeLV-FAIDS [the replication-competent common form (clone 61E) and the replication-defective variant (clone 61C)] in lymphocyte and leukocyte populations from infected cats. Micromagnetic separation of cell subsets, virus genome-specific PCR and flow cytometry were used to demonstrate the following sequence of events in infected animals: (i) very early replication of both 61E and 61C in CD4 T cells (provirus burden 0.2 to 1 copy/cell at 2-4 weeks post-infection); (ii) lower magnitude replication of both viruses in CD8 T cells and B cells during this initial phase of infection; (iii) plateauing of CD4 cell virus burden accompanied by escalation in CD8 and B cell provirus burdens after 4 weeks; (iv) extensive infection of haemopoietic and circulating myeloid cells. FeLV-FAIDS 61E and 61C replication kinetics and lymphocyte tropisms were similar in blood and lymph nodes, where provirus burdens ranged from 0.15 to 1.0 copy/cell. Moreover, virus infection was productive; 8-48 percent of blood lymphocytes, 35-81 percent of node lymphocytes and 53-98 percent of bone marrow cells expressed FeLV capsid antigen (p27 Gag). These findings suggest that the immunosuppressive potency of FeLV-FAIDS reflects the unique cytopathicity rather than unique cytotropism of its 61C (versus 61E) component.

Animals↗
PubMed1996

Generation and role of defective proviruses in cytopathic feline leukemia virus (FeLV-FAIDS) infections.

Cytopathic feline leukemia virus (FeLV) infections of feline T-cell line (FeT-cell) cultures led to the accumulation and maintenance of threefold more proviruses with deletions within the polymerase gene (pol) than minimally cytopathic FeLV infections. Over 60% of the viral DNA from cytopathic infections bore deletions in pol. Characterization of DNA sequences adjoining the deletions revealed that the junctions were most often flanked by RNA splice donor and acceptor consensus motifs. A thymidine-to-cytidine mutation introduced at the +2 position of one RNA splice donor-like motif inhibited formation of the two most prevalent viral DNA species with deletions, confirming the origin of many proviruses with deletions from reverse transcription of aberrantly spliced viral RNA species. An example of deletion by misalignment was also characterized. Viral inocula obtained from cells recovered after cytopathic infections were attenuated in their ability to cause cytopathic effects (CPE) and were able to confer superinfection resistance to naïve FeT-cells, despite maintaining envelope gene (env) sequences with full cytopathic potential. This suggested that viral genomes with deletions, rather than being required for cytopathicity, play a role in protecting cells from CPE. Indeed, expression of a molecularly cloned provirus bearing one of the characterized deletions attenuated CPE in FeT-cells caused by superinfecting cytopathic virus.

Animals↗
PubMed1996

Simian immunodeficiency virus-specific cytotoxic T-lymphocyte induction through DNA vaccination of rhesus monkeys.

In view of the growing evidence that virus-specific cytotoxic T lymphocytes (CTL) play an important role in containing the early spread of human immunodeficiency virus type 1 (HIV-1) in infected individuals, novel vaccine strategies capable of eliciting HIV-1-specific CTL are being pursued in attempts to create an effective AIDS vaccine. We have used the simian immunodeficiency virus of macaques (SIVmac)/rhesus monkey model to explore the induction of AIDS virus-specific CTL responses by DNA vaccination. We found that the inoculation of rhesus monkeys with plasmid DNA encoding SIVmac Env and Gag elicited a persisting SIVmac-specific memory CTL response. These CTL were CD8+ and major histocompatibility complex class I restricted. These studies provide evidence for the potential utility of DNA inoculation as an approach to an HIV-1 vaccine.

Animals↗
PubMed1996

Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy.

Multidrug-resistant human immunodeficiency virus type 1 (HIV-1) strains with reverse transcriptase (RT) mutations at codons A62-->V, V75-->I, F77-->L, F116-->Y, and Q151-->M have been reported in patients receiving combination therapy with zidovudine (AZT) and didanosine (ddI). Infectious clones with each mutation alone, all five mutations together, and various combinations of mutations were created by site-directed mutagenesis. Mutation Q151-->M conferred partial resistance to AZT, ddI, zalcitibine, and stavudine, whereas a combination of four mutations conferred increased resistance to AZT, ddI, zalcitibine, and stavudine. The positions of residues 75, 77, and 151 in the three-dimensional crystal structure of HIV-1 RT suggest that these residues may affect the ability of the enzyme to discriminate between deoxynucleoside triphosphates and nucleoside analog RT inhibitors. Replication experiments showed that clones with mutation F77-->L but without V75-->I (HIV-1(77), HIV-1(77,151), and HIV-1(77,116,151) had attenuated growth compared with that of the original HIV-1NL4-3 strain and strains containing mutations at both positions 75 and 77 (HIV-1(75,77,151) and HIV-1(75,77,116,15)). Sequence analysis of viral RNA and proviral DNA from several patients indicated that RT mutations developed in a sequential and cumulative pattern over the course of a 2- to 4-year observation period. The present results suggest that drug resistance and viral replicative capacity both may play a role in selection of HIV-1 RT mutations.

Animals↗
PubMed1996

Protection against lethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by a gp120 subunit vaccine.

Infection of pigtail macaques with SIVsmmPBj14, biological clone 3 (SIV-PBj14-bc13), produces an acute and usually fatal shock-like syndrome 7 to 14 days after infection. We used this simian immunodeficiency virus (SIV) model as a rapid and rigorous challenge to evaluate the efficacy of two SIV Env vaccine strategies. Groups of four pigtail macaques were immunized four times over a 25-week span with either a recombinant Semliki Forest virus expressing the SIV-PBj14 Env gp160 (SFV-SIVgp160) or purified recombinant SIV-PBj14 gp120 (rgp120) in SBN-1 adjuvant. Antibody titers to SIV Env developed in all immunized animals (mean peak titers prior to challenge, 1:1,700 for SFV-SIV gp 160 and 1:10,500 for rgp120), but neither neutralizing antibodies nor SIV-specific T-cell proliferative responses were detectable in any of the vaccinees. All macaques were challenged with a 100% infectious, 75% fatal dose of SIV-PBj14-bc13 at week 26. Three of four control animals died of acute SIV-PBj14 syndrome on days 12 and 13. By contrast, all four SFV-SIVgp160-immunized animals and three of the four rgp120-immunized animals were protected from lethal disease. While all virus-challenged animals became infected, symptoms of the SIV-PBj14 syndrome were more severe in controls than in vaccinees. Mean virus titers in plasma at 13 days postchallenge were approximately 10-fold lower in vaccinated than control animals. However, there was no apparent correlation between survival and levels of peripheral blood mononuclear cell-associated culturable virus, provirus load, or any antiviral immunologic parameter examined. The results indicate that while immunization with SFV-SIVgp160 and rgp120 did not protect against virus infection, these Env vaccines did lower the virus load in plasma and protect against the lethal SIV-PBj14 challenge.

Animals↗
PubMed1996

Simian immunodeficiency virus DNA vaccine trial in macaques.

An experimental vaccine consisting of five DNA plasmids expressing different combinations and forms of simian immunodeficiency virus-macaque (SIVmac) proteins has been evaluated for the ability to protect against a highly pathogenic uncloned SIVmac251 challenge. One vaccine plasmid encoded nonreplicating SIVmac239 virus particles. The other four plasmids encoded secreted forms of the envelope glycoproteins of two T-cell-tropic relatives (SIVmac239 and SIVmac251) and one monocyte/macrophage-tropic relative (SIVmac316) of the uncloned challenge virus. Rhesus macaques were inoculated with DNA at 1 and 3, 11 and 13, and 21 and 23 weeks. Four macaques were inoculated intravenously, intramuscularly, and by gene gun inoculations. Three received only gene gun inoculations. Two control monkeys were inoculated with control plasmids by all three routes of inoculation. Neutralizing antibody titers of 1:216 to 1:768 were present in all of the vaccinated monkeys after the second cluster of inoculations. These titers were transient, were not boosted by the third cluster of inoculations, and had fallen to 1:24 to 1:72 by the time of challenge. Cytotoxic T-cell activity for Env was also raised in all of the vaccinated animals. The temporal appearance of cytotoxic T cells was similar to that of antibody. However, while antibody responses fell with time, cytotoxic T-cell responses persisted. The SIVmac251 challenge was administered intravenously at 2 weeks following the last immunization. The DNA immunizations did not prevent infection or protect against CD4+ cell loss. Long-term chronic levels of infection were similar in the vaccinated and control animals, with 1 in 10,000 to 1 in 100,000 peripheral blood cells carrying infectious virus. However, viral loads were reduced to the chronic level over a shorter period of time in the vaccinated groups (6 weeks) than in the control group (12 weeks). Thus, the DNA vaccine raised both neutralizing antibody and cytotoxic T-lymphocyte responses and provided some attenuation of the acute phase of infection, but it did not prevent the loss of CD4+ cells.

Animals↗
PubMed1996

Maintaining the integrity of human immunodeficiency virus sequence databases.

Human immunodeficiency virus type 1 (HIV-1) sequences are accumulating in the literature at a rapid pace. For this ever-expanding resource to be maximally useful, it is critical that researchers strive to maintain a high level of quality assurance, both in experimental design and conduct and in analyses. Here we present detailed analyses of problematic sets of HIV-1 sequences in the database that include sequence anomalies suggestive of mislabeling or sample contamination problems. These data are examined in the context of currently available HIV-1 sequence information to provide an example of how to identify potentially flawed data. Indicators of potential problems with sequences are (i) sequences that are nearly identical that are supposed to be derived from unlinked individuals and that are markedly distinct from other sequences from the putative source or (ii) sequences that are nearly identical to those of laboratory strains. We provide an outline of methods that researchers can use to perform preliminary laboratory and computational analyses that could help identify problematic data and thus help ensure the integrity of sequence databases.

Base Sequence↗