Roger L. Shapiro

Adaptation of HIV-1 to human leukocyte antigen class I

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host–pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8+ T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8+ T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128–135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8+ T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Botulism in the united states : A clinical and epidemiologic review

Botulism is a neuroparalytic illness caused by a neurotoxin produced from the anaerobic, spore-forming bacterium Clostridium botulinum [1]. Botulism was recognized as sausage poisoning during the 18th and 19th centuries [2], and the pathogenesis of disease was first described by van Ermengem in 1897 after his investigation of a large outbreak in Ellezelles, Belgium [3]. Because botulinum toxin is so lethal, intensive surveillance and control measures have been mandated in the United States. However, prompt recognition and treatment of botulism by clinicians remain a critical component of surveillance and are the most important steps in reducing rates of death from this disease. Botulism outbreaks are a public health emergency that require rapid recognition to prevent additional cases and to effectively treat patients with mechanical ventilation and early administration of antitoxin. In the event of terrorist use of botulinum toxin, clinicians would also be the first to recognize and treat casualties of intentional botulism poisoning. In this report, we provide a clinical overview of botulism and describe the U.S. Botulism Surveillance System. The Organism Clostridium botulinum is classified as a single species but consists of at least three genetically distinguishable groups of organisms. These are alike in their abilities to produce neurotoxins with similar pharmacologic activities [4] but diverse serologic properties (toxin types A, B, C, D, E, F, and G). Human botulism is primarily caused by the strains of C. botulinum that produce toxin types A, B, and E. Neurotoxigenic strains of C. baratii [5, 6] (which produce type F toxin) and C. butyricum [7] (which produce type E toxin) also have been implicated in human botulism. Strains of C. botulinum that produce type C or type D toxin for the most part cause botulism only in nonhuman species. These neurotoxigenic organisms are anaerobic, gram-positive, spore-forming bacilli and are commonly found in soils throughout the world. Clostridium botulinum organisms cause food poisoning because the heat-resistant spores survive food preservation methods that kill nonsporulating organisms; they subsequently produce a potent neurotoxin under anaerobic, low-acid (pH > 4.6), and low solute conditions [8]. The toxins affect a broad range of vertebrate species, but the evolutionary utility of toxin production to the bacterial host organisms is unclear. The Toxin The seven recognized types of botulinum neurotoxins (types A through G) are distinguished by neutralization of biological activity with type-specific serologic reagents. These types are defined by the International Standards for Clostridium botulinum Antitoxin [9]. The toxins of all types consist of a 100-kd heavy chain joined to a 50-kd light chain by a disulfide bond [10]. After absorption into the bloodstream, botulinum toxin binds irreversibly to the presynaptic nerve endings of the peripheral nervous system and cranial nerves, where it inhibits the release of acetylcholine (Figure 1). The mechanism involves binding to a toxin receptor on the nerve cell membrane at the neuromuscular junction, internalization of a portion (the catalytic portion residing in the light chain) of the toxin molecule [11], and cleavage of protein components of the neuroexocytosis apparatus within the cell [12]. Figure 1. Schematic representation of the action of botulinum toxin (BT) on a neuromuscular junction. Botulinum neurotoxin is considered the most potent lethal substance known. It is 15 000 to 100 000 times more toxic than sarin, the potent organophosphate nerve agent used in a terrorist attack in the subway system in Tokyo [13]. The nucleotide sequences for all seven toxin types have been sequenced [14-22]. Epidemiology Four clinical forms of botulism occur in humans: foodborne botulism; wound botulism; infant botulism (infant intestinal colonization); and, rarely, adult infectious botulism (adult intestinal colonization). Studies in monkeys indicate that, if aerosolized, botulinum toxin also can be absorbed through the lungs [23]; this could occur in the case of a terrorist attack. From 1973 through 1996 in the United States, 724 cases of foodborne botulism (median, 24 cases annually [range, 8 to 86 cases]), 103 cases of wound botulism (median, 3 cases annually [range, 0 to 25 cases]), 1444 cases of infant botulism (median, 71 cases annually [range, 0 to 99 cases]), and 39 cases of botulism of undetermined type were reported to the Centers for Disease Control and Prevention (CDC) (Figure 2) (CDC. Unpublished data). In the United States, approximately half of the cases of foodborne botulism are caused by toxin type A; the remaining foodborne cases are almost equally divided between toxins type E and type B [24]. Among cases of infant botulism, approximately half are caused by toxin type A and half by toxin type B; among cases of wound botulism, approximately 80% are caused by toxin type A and 20% by toxin type B (CDC. Unpublished data). In the United States, type A botulism is most common west of the Mississippi River, and type B is most common east of the Mississippi River [25]. Type E outbreaks are most common in Alaska [26, 27]. Figure 2. Annual incidence of botulism in the United States, 1973 to 1996. Important changes in the epidemiology of botulism have emerged in the past few decades. Recently identified vehicles for foodborne botulism include homemade salsa [24], baked potatoes sealed in aluminum foil [28], cheese sauce [29], sauteed onions held under a layer of butter [30], garlic in oil [31], and traditionally prepared salted or fermented fish [26] (Table 1). From 1976 through 1984, restaurant-associated outbreaks accounted for a large proportion of botulism cases (42%), although only 4% of all outbreaks were restaurant-associated [32]. The largest of these outbreaks were caused by jalapeno peppers in Michigan in 1977, potato salad in New Mexico in 1978, sauteed onions in Illinois in 1983, and skordalia made with baked potatoes in Texas in 1994 [33]. Table 1. Vehicles Associated with Foodborne Botulism In 1995 and 1996, the occurrence of wound botulism increased [34], with a total of 42 cases (CDC. Unpublished data). Most of these cases occurred among heroin users in California who injected the drug subcutaneously. Although it is unclear what factors contributed to this epidemic, a shift to the use of black tar heroin produced in Mexico may have played a role [35]. Purified botulinum toxin is used to treat various medical conditions, such as strabismus, blepharospasm, torticollis, oromandibular dystonia, spasmodic dysphonia, and achalasia. Systemic symptoms of botulism-like illness have been reported after therapeutic administration of botulinum toxin [36] but are unlikely to have resulted from this procedure. It is estimated that for most patients, at least 10 times the treatment dose would be required to enter the circulation for systemic symptoms to result ([37]; CDC. Unpublished data). The potential for intentional poisoning with botulinum toxin has come into clearer focus in recent years. As many as 17 countries are suspected to include or to be developing biological agents in their offensive weapons programs [38]. Botulinum toxin often is one of these agents because it is relatively easy to produce and is highly lethal in small quantities. In August 1995, Iraq revealed that during the Persian Gulf War, 11 200 L of botulinum toxin preparation was loaded into specially designed SCUD missile warheads [39]. In addition, before the Aum Shinrikyo used sarin in the 1995 terrorist attack on the Tokyo subway system, the cult had produced botulinum toxin [40]. Clinical Features Foodborne Botulism Foodborne botulism is caused by ingestion of preformed toxin produced in food by C. botulinum. The most frequent source is home-canned foods, in which spores that survive an inadequate cooking and canning process germinate, reproduce, and produce toxin in the anaerobic environment of the canned food. In the event of intentional foodborne poisoning with botulinum toxin, the signs and symptoms developing after ingestion would probably resemble those of naturally occurring foodborne botulism. If aerosolized toxin was inhaled, the incubation period might be slightly longer [23], and gastrointestinal symptoms might not occur. The clinical syndrome of foodborne botulism is dominated by neurologic symptoms and signs resulting from a toxin-induced blockade of the voluntary motor and autonomic cholinergic junctions (Table 2). Although the syndrome is similar for each toxin type, type A toxin has been associated with more severe disease and a higher fatality rate than type B or type E toxin [41]. Symptoms from any toxin type may range from subtle motor weakness or cranial nerve palsies to rapid respiratory arrest. The initial symptoms of foodborne botulism may be gastrointestinal and can include nausea, vomiting, abdominal cramps, or diarrhea; after the onset of neurologic symptoms, constipation is more typical. Dry mouth, blurred vision, and diplopia are usually the earliest neurologic symptoms. These initial symptoms may be followed by dysphonia, dysarthria, dysphagia, and peripheral muscle weakness. Symmetric descending paralysis is characteristic of botulism; paralysis begins with the cranial nerves, then affects the upper extremities, the respiratory muscles, and, finally, the lower extremities in a proximal-to-distal pattern. Onset usually occurs 18 to 36 hours after exposure (range, 6 hours to 8 days) [42]. In severe cases, extensive respiratory muscle paralysis leads to ventilatory failure and death unless supportive care is provided. Patients have required ventilatory support for up to 7 months before the return of muscular function, but ventilatory support is most commonly needed for 2 to 8 weeks [43]. Table 2. Commonly Reported Clinical Symptoms and Physical Findings in Botulism* Clinical recovery generally occurs over weeks to m

The Role of Gulf Coast Oysters Harvested in Warmer Months in Vibrio vulnificus Infections in the United States, 1988–1996

Vibrio vulnificus infections are highly lethal and associated with consumption of raw shellfish and exposure of wounds to seawater. V. vulnificus infections were reported to the Centers for Disease Control and Prevention from 23 states. For primary septicemia infections, oyster trace-backs were performed and water temperature data obtained at harvesting sites. Between 1988 and 1996, 422 infections were reported; 45% were wound infections, 43% primary septicemia, 5% gastroenteritis, and 7% from undetermined exposure. Eighty-six percent of patients were male, and 96% with primary septicemia consumed raw oysters. Sixty-one percent with primary septicemia died; underlying liver disease was associated with fatal outcome. All trace-backs with complete information implicated oysters harvested in the Gulf of Mexico; 89% were harvested in water >22 degrees C, the mean annual temperature at the harvesting sites (P < .0001). Control measures should focus on the increased risk from oysters harvested from the Gulf of Mexico during warm months as well as education about host susceptibility factors.

Highly Active Antiretroviral Therapy and Adverse Birth Outcomes Among HIV-Infected Women in Botswana

BACKGROUND It is unknown whether adverse birth outcomes are associated with maternal highly active antiretroviral therapy (HAART) in pregnancy, particularly in resource-limited settings. METHODS We abstracted obstetrical records at 6 sites in Botswana for 24 months. Outcomes included stillbirths (SBs), preterm delivery (PTD), small for gestational age (SGA), and neonatal death (NND). Among human immunodeficiency virus (HIV)-infected women, comparisons were limited to HAART exposure status at conception, and those with similar opportunities for outcomes. Comparisons were adjusted for CD4(+) lymphocyte cell count. RESULTS Of 33,148 women, 32,113 (97%) were tested for HIV, of whom 9504 (30%) were HIV infected. Maternal HIV was significantly associated with SB, PTD, SGA, and NND. Compared with all other HIV-infected women, those continuing HAART from before pregnancy had higher odds of PTD (adjusted odds ratio [AOR], 1.2; 95% confidence interval [CI], 1.1, 1.4), SGA (AOR, 1.8; 95% CI, 1.6, 2.1) and SB (AOR, 1.5; 95% CI, 1.2, 1.8). Among women initiating antiretroviral therapy in pregnancy, HAART use (vs zidovudine) was associated with higher odds of PTD (AOR, 1.4; 95% CI, 1.2, 1.8), SGA (AOR, 1.5; 95% CI, 1.2, 1.9), and SB (AOR, 2.5; 95% CI, 1.6, 3.9). Low CD4(+) was independently associated with SB and SGA, and maternal hypertension during pregnancy with PTD, SGA, and SB. CONCLUSIONS HAART receipt during pregnancy was associated with increased PTD, SGA, and SB.

Selection bias at the heterosexual HIV-1 transmission bottleneck

Introduction Heterosexual HIV-1 transmission is an inefficient process with rates reported at <1% per unprotected sexual exposure. When transmission occurs, systemic infection is typically established by a single genetic variant, taken from the swarm of genetically distinct viruses circulating in the donor. Whether that founder virus represents a chance event or was systematically favored is unclear. Our work has tested a central hypothesis that founder virus selection is biased toward certain genetic characteristics. Fitter viruses (red) are favored more in woman-to-man (bottom curve) than in man-to-woman (top curve) transmission. The probability that a majority donor amino acid variant is transmitted is a function of relative fitness, here estimated by the frequency of the variant in the Zambian population. Even residues common in the population are less likely to be transmitted to healthy men than to women, indicative of higher selection bias in woman-to-man transmission. Rationale If HIV-1 transmission involves selection for viruses with certain favorable characteristics, then such advantages should emerge as statistical biases when viewed across many viral loci in many transmitting partners. We therefore identified 137 Zambian heterosexual transmission pairs, for whom plasma samples were available for both the donor and recipient partner soon after transmission, and compared the viral sequences obtained from each partner to identify features that predicted whether the majority amino acid observed at any particular position in the donor was transmitted. We focused attention on two features: viral genetic characteristics that correlate with viral fitness, and clinical factors that influence transmission. Statistical modeling indicates that the former will be favored for transmission, while the latter will nullify this relative advantage. Results We observed a highly significant selection bias that favors the transmission of amino acids associated with increased fitness. These features included the frequency of the amino acid in the study cohort, the relative advantage of the amino acid with respect to the stability of the protein, and features related to immune escape and compensation. This selection bias was reduced in couples with high risk of transmission. In particular, significantly less selection bias was observed in men with genital inflammation and in women (regardless of inflammation status), compared to healthy men, suggesting a more permissive environment in the female than male genital tract. Consistent with this observation, viruses transmitted to women were characterized by lower predicted fitness than those in men. The presence of amino acids favored during transmission predicted which individual virus within a donor was transmitted to their partner, while chronically infected individuals with viral populations characterized by a predominance of these amino acids were more likely to transmit to their partners. Conclusion These data highlight the clear selection biases that benefit fitter viruses during transmission in the context of a stochastic process. That such biases exist, and are tempered by certain risk factors, suggests that transmission is frequently characterized by many abortive transmission events in which some target cells are nonproductively infected. Moreover, for efficient transmission, some changes that favored survival in the transmitting partner are frequently discarded, resulting in overall slower evolution of HIV-1 in the population. Paradoxically, by increasing the selection bias at the transmission bottleneck, reduction of susceptibility may increase the expected fitness of breakthrough viruses that establish infection and may therefore worsen the prognosis for the newly infected partner. Conversely, preventive or therapeutic approaches that weaken the virus may reduce overall transmission rates via a mechanism that is independent from the quantity of circulating virus, and may therefore provide long-term benefits to the recipient if transmission does occur. HIV needs to be fit to transmit Although you might not think it, its hard to catch HIV. Less than 1% of unprotected sexual exposures result in infection. What then leads to transmission? Carlson et al. determined the amino acid sequence of viruses infecting 137 Zambian heterosexual couples in which one partner infected the other (see the Perspective by Joseph and Swanstrom). The authors then used statistical modeling and found that transmitted viruses are typically the most evolutionarily fit. That is, compared to other viral variants in the infected person, the transmitted virus most closely matches the most common viral sequence found in the Zambian population. Science, this issue 10.1126/science.1254031; see also p. 136 An analysis of discordant couples reveals that transmitted HIV-1 viruses are typically the most evolutionarily fit. [Also see Perspective by Joseph and Swanstrom] Heterosexual transmission of HIV-1 typically results in one genetic variant establishing systemic infection. We compared, for 137 linked transmission pairs, the amino acid sequences encoded by non-envelope genes of viruses in both partners and demonstrate a selection bias for transmission of residues that are predicted to confer increased in vivo fitness on viruses in the newly infected, immunologically naïve recipient. Although tempered by transmission risk factors, such as donor viral load, genital inflammation, and recipient gender, this selection bias provides an overall transmission advantage for viral quasispecies that are dominated by viruses with high in vivo fitness. Thus, preventative or therapeutic approaches that even marginally reduce viral fitness may lower the overall transmission rates and offer long-term benefits even upon successful transmission.

Male circumcision: an acceptable strategy for HIV prevention in Botswana.

Background: Male circumcision is known to reduce the risk of acquiring HIV, but few studies have been performed to assess its acceptability among either children or adults in sub-Saharan Africa. Methods: We conducted a cross sectional survey in nine geographically representative locations in Botswana to determine the acceptability of male circumcision in the country, as well as the preferred age and setting for male circumcision. Interviews were conducted using standardised questionnaires both before and after an informational session outlining the risks and benefits of male circumcision. Results: Among 605 people surveyed, the median age was 29 years (range 18–74 years), 52% were male, and >15 ethnicities were represented. Before the informational session, 408 (68%) responded that they would definitely or probably circumcise a male child if circumcision was offered free of charge in a hospital setting; this number increased to 542 (89%) after the informational session. Among 238 uncircumcised men, 145 (61%) stated that they would definitely or probably get circumcised themselves if it were offered free of charge in a hospital setting; this increased to 192 (81%) after the informational session. In a multivariate analysis of all participants, people with children were more likely to favour circumcision than people without children (adjusted odds ratio 1.8, 95% CI 1.0 to 3.4). Most participants (55%) felt that the ideal age for circumcision is before 6 years, and 90% of participants felt that circumcision should be performed in the hospital setting. Conclusions: Male circumcision appears to be highly acceptable in Botswana. The option for safe circumcision should be made available to parents in Botswana for their male children. Circumcision might also be an acceptable option for adults and adolescents, if its efficacy as an HIV prevention strategy among sexually active people is supported by clinical trials.

Increased Risk of Preterm Delivery Among HIV-Infected Women Randomized to Protease Versus Nucleoside Reverse Transcriptase Inhibitor-Based HAART During Pregnancy

BACKGROUND Protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) use in pregnancy has been associated with preterm deliveries in some observational studies. METHODS HIV-infected, HAART-naive pregnant women with CD4+ counts ≥200 cells/mm(3) were randomized between 26 and 34 weeks gestation to lopinavir/ritonavir/zidovudine/lamivudine (PI group) or abacavir/zidovudine/lamivudine (NRTI group) in a clinical trial to prevent mother-to-child HIV transmission. Risk factors for preterm delivery (<37 weeks) and differences by randomization arm were evaluated for live infants by logistic regression. RESULTS Preterm delivery rates were higher among 267 women in the PI group than 263 women in the NRTI group (21.4% vs 11.8%, P = .003). PI-based HAART was the most significant risk factor for preterm delivery [odds ratio = 2.03, 95% confidence interval 1.26-3.27, P = .004]. Mean change in maternal body mass index (BMI) 1 month after HAART initiation was lower in the PI group (P < .001); however, this was not significantly associated with preterm delivery. Neither infant hospitalizations nor mortality through 6 months of life differed by maternal regimen. CONCLUSIONS PI-based HAART was associated with increased preterm delivery but not increased infant hospitalizations or mortality in a clinical trial setting. The association between PI use and lower increase in BMI in late pregnancy warrants further study.

Infant Morbidity, Mortality, and Breast Milk Immunologic Profiles among Breast-Feeding HIV-Infected and HIV-Uninfected Women in Botswana

BACKGROUND Infants of human immunodeficiency virus (HIV)-infected women have high mortality, but the immunologic integrity and protection afforded by the breast milk of HIV-infected women is unknown. METHODS We determined morbidity and mortality by 24 months among breast-fed infants of 588 HIV-infected and 137 HIV-uninfected women followed-up in a clinical trial in Botswana. A matched case-control study compared clinical, behavioral, and breast milk immunologic parameters among 120 HIV-infected women by infant outcome. Breast milk factors were also compared between HIV-infected and HIV-uninfected women. RESULTS Twenty-four-month mortality was 29.5% among HIV-infected infants, 6.7% among HIV-exposed uninfected infants, and 1.6% among HIV-unexposed infants. No differences were detected in breast milk immunologic profiles of HIV-infected women whose infants were either ill or well. Discontinuation of breast-feeding was the strongest predictor of illness (P<.001). Levels in breast milk of pathogen-specific immunoglobulin (Ig) G and IgA to Haemophilus influenzae, Campylobacter jejuni, Helicobacter pylori, Streptococcus pneumoniae, and innate immune factors were not lower among HIV-infected women than among HIV-uninfected women. CONCLUSIONS Mortality was higher among HIV-infected and HIV-exposed infants than among HIV-unexposed infants. However, the immunologic profiles of breast milk among HIV-infected women were intact, and discontinuation of breast-feeding was the primary risk for infant morbidity. Thus, the breast milk of HIV-infected women may confer protection against common infant pathogens. TRIAL REGISTRATION (ClinicalTrials.Gov) identifiers: NCT00197691 and NCT00197652.

Antiretroviral Concentrations in Breast-Feeding Infants of Women in Botswana Receiving Antiretroviral Treatment

BACKGROUND The magnitude of infant antiretroviral (ARV) exposure from breast milk is unknown. METHODS We measured concentrations of nevirapine, lamivudine, and zidovudine in serum and whole breast milk from human immunodeficiency virus type 1 (HIV-1)-infected women in Botswana receiving ARV treatment and serum from their uninfected, breast-feeding infants. RESULTS Twenty mother-infant pairs were enrolled. Maternal serum concentrations of nevirapine were high (median, 9534 ng/mL at a median of 4 h after nevirapine ingestion). Median breast-milk concentrations of nevirapine, lamivudine, and zidovudine were 0.67, 3.34, and 3.21 times, respectively, those in maternal serum. The median infant serum concentration of nevirapine was 971 ng/mL, at least 40 times the 50% inhibitory concentration and similar to peak concentrations after a single 2-mg/kg dose of nevirapine. The median infant serum concentration of lamivudine was 28 ng/mL, and the median infant serum concentration of zidovudine was 123 ng/mL, but infants were also receiving zidovudine prophylaxis. CONCLUSIONS HIV-1 inhibitory concentrations of nevirapine are achieved in breast-feeding infants of mothers receiving these ARVs, exposing infants to the potential for beneficial and adverse effects of nevirapine ingestion. Further study is needed to understand the impact of maternal ARV treatment on breast-feeding HIV-1 transmission, infant toxicity, and HIV-1 resistance mutations among infected infants.

Highly Active Antiretroviral Therapy Started during Pregnancy or Postpartum Suppresses HIV-1 RNA, but Not DNA, in Breast Milk

BACKGROUND The ability of highly active antiretroviral therapy (HAART) to reduce human immunodeficiency virus type 1 (HIV-1) RNA and DNA in breast milk has not been described. METHODS We compared breast-milk HIV-1 RNA and DNA loads of women in Botswana who received HAART (nevirapine, lamivudine, and zidovudine) and women who did not receive HAART. RESULTS Women in the HAART group received treatment for a median of 98 days (range, 67-222 days) at the time of breast-milk sampling; 23 (88%) of 26 had whole breast-milk HIV-1 RNA loads <50 copies/mL, compared with 9 (36%) of 25 women who did not receive HAART (P=.0001). This finding remained significant in a multivariate logistic-regression model (P = .0006). The whole-milk HIV-1 DNA load was unaffected by HAART. Of women who received HAART, 13 (50%) of 26 had HIV-1 DNA loads <10 copies/10(6) cells, compared with 15 (65%) of 23 who did not receive HAART (P = .39). CONCLUSIONS HAART suppressed cell-free HIV-1 RNA in breast milk and may therefore reduce mother-to-child transmission (MTCT) of HIV-1 via breast-feeding. However, HAART initiated during pregnancy or early after delivery had no apparent effect on cell-associated HIV-1 DNA loads in breast milk. Clinical trials to determine MTCT among breast-feeding women receiving HAART are needed.