Primary HIV-1 infection in Zurich: molecular studies of transmission biology and epidemiology

Mit der antiretroviralen Therapie (ART) konnte ein deutlicher Erfolg bei der Behandlung von HIV-infizierten Patienten erzielt werden. Bis jetzt ist es jedoch unmöglich mit antiretroviralen Medikamenten HIV-1 Patienten zu heilen. In den westlichen Ländern gab es in den letzten Jahren eine erneute Zunahme von HIV-1 Diagnosen, besonders unter homosexuellen Männern. In den Entwicklungsländern ist es unwahrscheinlich, dass durch ART die Epidemie von HIV-1 unter Kontrolle gebracht werden kann. Daher ist die Erforschung eines sicheren und wirksamen HIV- Impfstoffes von entscheidender Bedeutung um die Epidemie zu stoppen. Trotz grosser Bemühungen sind empirische Ansätze zur Entwicklung von HIV-1 Impfstoffen bisher fehlgeschlagen. Zur grössten Hürde in der Impfstoffentwicklung gehört die enorme Variabilität der HI-Viren. Bei der HIV-1 Ansteckung werden jedoch nur eine oder wenige virale Varianten auf den Empfänger übertragen. Dieser genetische Flaschenhals könnte eine Achillesferse des HI-Virus darstellen. Beim Menschen wird der biologische Übertragungsmechanismus jedoch nur bruchstückhaft verstanden. Vertieftes Wissen über den Mechanismus des genetischen Flaschenhalses, sowie über die Eigenschaften der übertragenen Viren könnten wesentliche Schritte zu einem effektiven Impfstoff sein. Die virale Diversität ist einerseits ein Hindernis in der Impfstoffentwicklung, andererseits ermöglicht sie auch phylogentische Studien des viralen Genoms. Die phylogenetische Analyse der viralen Sequenzen während der primären Phase der HIV-1 Infektion ermöglicht die frühen Wirt-Virus Interaktionen zu studieren, welches sonst nur im Tiermodell möglich ist. In Kapitel 1 wird die Komplexität der übertragenen HIV-1-Populationen untersucht und nach Risikofaktoren geforscht, welche die Transmission von mehreren Viren ermöglicht. Unsere Ergebnisse legen nahe, dass die Übertragung von mehreren genetischen Varianten nicht ausschliesslich von den Eigenschaften der Schleimhaut abhängig ist, wie dies in kürzlich publizierten Studien propagiert wurde. Die Übertragung von Virenstämmen, die den Co-Rezeptor CXCR4 benützen, ist ein seltenes Ereignis. Vorsicht ist geboten bei der Vorhersage der Co-Rezeptor-Nutzung durch genotypische Algorithmen, denn die Konkordanz dieser bioinformatischen Werkzeuge ist begrenzt. Im Jahr 2009 traten weltweit schätzungsweise 2,6 Millionen neue Fälle von HIV-1 Infektion auf. Neuere Daten deuten auf eine hohe HIV-1-Übertragungsrate von Personen mit primärer HIV-1 Infektion. Kapitel 2 beschreibt die Transmissionsdynamik während der akuten Infektion über den Zeitraum der ART und in der chronischen Phase nach dem Absetzen der ART. Innerhalb phylogenetischer Cluster suchen wir mit Hilfe longitudinaler klinischer Daten nach Übertragungsmustern. Eine große Zahl von Neuinfektionen stammt von Patienten, welche die ART gestoppt hatten. Dies weist darauf hin, dass aktuelle präventive Bemühungen unzureichend sind. Diese Befunde sprechen für eine frühzeitige, kontinuierliche ART um die Ausbreitung von HIV-1, insbesondere bei Personen mit häufiger sexueller Aktivität, zu reduzieren. Der Erfolg der ART in einem einzelnen Patienten wird durch die Messung viraler RNA im Blut überwacht. Aktuelle kommerziell erhältliche „Assays“ haben eine Nachweisgrenze zwischen 20 und 50 viraler RNA-Kopien pro ml Blut. In epidemiologischen Studien bezüglich des Übertragungsrisikos unter ART ist es nützlich, wenn auch einzelne replizierende Viren nachgewiesen werden können. Das HI-Virus verändert sich in hohem Masse. Daher müssen molekulare Nachweisverfahren eine breite Palette von Virenstämme erkennen. Kapitel 3 dieser Dissertation beschreibt eine Methode um phylogenetisch diverse Viren, wie HIV-1, mit einer hohen Sensivität zu detektieren. Nach der primären HIV-Infektion nivelliert sich die Viruslast (VL) eines Patienten auf einem bestimmten Niveau ein. Dieser Wert, genannt viraler „setpoint“, kann mehr als 1000-fach zwischen Patienten variieren und ist ein Marker für den Krankheitsverlauf. Der virale „setpoint“ wird durch genetische Komponenten des Wirtes und des Virus bestimmt. In Kapitel 4 wird mit der Anwendung von „machine learning tools“ versucht, eine Signatur im viralen Genom zu identifizieren, welche mit einem tiefen viralen „setpoint“ assoziiert ist. Unsere Ergebnisse legen nahe, dass Patienten mit der Präsenz von Viren, welchen das Aminosäure-Muster 268E/358T fehlt, eine VL von > 5000 bei Studienbeginn aufzeigen und mit geringer Wahrscheinlichkeit das Virus nach Absetzen der ART kontrollieren können. Dieses Aminosäure-Muster in HIV-1 gp120 könnte sich in vivo negativ auf die virale Fitness auswirken. HIV-1-Infektionen werden von einer oder wenigen viralen Varianten oder „Quasispezies“ initiiert. Anschließend nimmt mit fortschreitender Erkrankung die virale Diversität mehr oder weniger linear zu. So ist die virale genetische Vielfalt innerhalb eines Patienten potenziell informativ für das Alter der Infektion. Genotypische Resistenz-Tests werden heute routinemäßig bei Patienten mit einer HIV-1 Infektion durchgeführt. Die dazu verwendete Methode ist eine „bulk“- Sequenzierung der pol-Region. In Kapitel 5 analysieren wir, ob der Anteil der mehrdeutigen Nukleotide in den „bulk“-Sequenzen eines einzelnen Patienten Aufschluss über das Alter der Infektion aufgibt. Unsere Beobachtungen zeigen, dass der Anteil der mehrdeutigen Nukleotide mit einer Rate von 0,2% pro Jahr innerhalb der ersten 8 Jahre steigt. Darüber hinaus wird gezeigt, dass ein Anteil der mehrdeutigen Nukleotide von > 0,5% starke Beweise gegen eine frische Infektion liefert (Transmission < 1 Jahr vor der Probenahme). Zusammenfassend kann gesagt werden, dass phylogenetische Studien der viralen Sequenzen verwendet werden können, um komplexe Fragestellungen wie epidemiologische Studien und HIV-1 Übertragungsmechanismen zu erforschen. Phylogenetische Analysen können zur Identifizierung von Transmissions-Paaren verwendet werden und das Erkennen von Übertragungsmustern hilft dabei, Präventionsstrategien zu verbessern. Das genaue Verständnis der initialen Abläufe während einer HIV-1 Neuinfektion könnte entscheidend sein für die Entwicklung eines wirksamen Impfstoffes. Phylogenetische Analysen viraler Sequenzen werden zukünftig an Bedeutung gewinnen, da zurzeit neue Sequenzierungstechniken etabliert werden und die Menge an Verfügbaren Sequenzen sprunghaft Ansteigt. Summary Although a marked success of antiretroviral therapy (ART) in controlling human immunodeficiency virus type 1 (HIV-1) infection, ART fails to cure patients from this virus. In addition, in developed countries there is an increase in new diagnoses of HIV-1, particularly in men having sex with men (MSM). In regions where HIV-1 has hit hardest it is unlikely to eradicate the virus by ART. Therefore, a safe and effective HIV-1 vaccine is crucial to halt the epidemic. Empirical approaches to the development of HIV-1 vaccines have so far, despite huge efforts, not been successful. Among the major obstacles is the enormous genetic variability. An Achilles heel of HIV-1 may be the genetic bottleneck during HIV-1 transmission, in which only one or a few viral variants, quasispecies, from a complex viral population in the source are transmitted to the recipient. However, knowledge about HIV-1 transmission biology in humans is still insufficient. Understanding the mechanisms of the genetic bottleneck and knowledge about the characteristics of the transmitted viral strains may help to design an effective HIV-1 vaccine. Contrarily to the obstacle in vaccine design, viral diversity enables phylogenetic studies of viral sequences. The phylogenetic investigation of viral sequences obtained during the acute and recent phase of HIV-1 infection facilitate the possibility to study transmission and early host/virus interactions which is otherwise only possible in animal models In chapter 1 investigation of the complexity of transmitted HIV-1 populations early after infection, and assessing potential factors associated with transmission of heterogeneous viral populations has been done. Our findings suggest that transmission of complex virus founder populations may not depend solely on mucosal factors, as suggested so far by recently published work. In addition, transmission of clinically relevant CXCR4-using viral strains seems to remain a rare event and caution is warranted predicting coreceptor usage by genotypic algorithms alone, because concordance of those tools is still limited. In 2009, an estimated 2.6 million new cases of HIV-1 infection occurred. Recent data suggests high HIV-1 transmission rates caused by individuals with primary HIV-1 infection. Chapter 2 describes the study of transmission dynamics during the acute infection, during the aviremic phase over the period of ART, and during the chronic stage after cessation of ART. We investigated transmission patterns within phylogenetic clusters by using longitudinal clinical data. Large numbers of new infections originated from patients who stopped ART indicating that current preventive efforts are insufficient. These findings argue for early, continuous ART to reduce the spread of HIV-1, in particular in individuals with frequent sexual activity. The success of ART in an individual patient is monitored by measuring HIV RNA copy numbers in blood plasma. Current commercial available assays have a detection limit between 20 and 50 viral RNA copy numbers per ml blood. Regarding epidemiological studies of transmission events, also under therapy, it is of interest to also detect viral RNA at single copy sensitivity. In addition HIV-1 is continuously evolving at a high rate. Therefore molecular detection assays have to detect a wide range of viral strains. Chapter 3 of this dissertation describes a method to detect phylogenetically diverse viruses such as HIV-1 with single-copy sensitivity. After primary HIV-1 infection (PHI) each individual reaches a steady-state level of plasma viral load which is referred to as viral setpoint. These setpoints between individuals can vary more than 1000 fold and are surrogate markers for disease progression. HIV-1 RNA levels are influenced by genetic characteristics of both the host and the virus. In chapter 4 we aim at identifying common genetic viral signatures associated with virologic control by applying machine learning tools. Our results suggest that presence of virus lacking the amino acid pattern 268E/358T is associated with viral load (VL) >5000 at baseline of PHI and with low probability of spontaneous virologic control after cessation of ART. These residues in HIV-1 gp120 might affect in vivo HIV-1 fitness. HIV-1 infection is initiated by one or a few closely related viruses. Subsequently, viral diversity increases with disease progression. Thus, the viral genetic diversity within a patient is potentially informative about the age of infection. Genotypic resistance tests are routinely done in HIV-1 infected patients by bulk sequencing of the viral pol region. In chapter 5, we aimed if the fraction of ambiguous nucleotides (nt) within pol bulk sequences obtained from an individual patient is informative about the age of infection. We found that the fraction of ambiguous nts increased at a rate of 0.2% per year within the first 8 years. Additionally, we showed that a fraction of ambiguous nts of >0.5% provides strong evidence against a recent infection event <1 year prior to sampling. Taken together, phylogenetic studies of viral nucleotide sequences can be used to address complex aims such as epidemiological studies and HIV-1 transmission biology. Phylogenetic analysis can be used to detect transmission pairs and the knowledge about transmission dynamics can improve prevention strategies. Better understanding how HIV-1 establishes a new infection may turn out to be a prerequisite to design efficient HIV-1 vaccines. Additionally, in the future, the phylogenetic study of viral sequences will be of great importance because new sequencing techniques are currently developed, such as 454 sequencing, and the amount of available sequences will increase exponentially

Parcours de praticien et savoir historique : quelles histoires pour un médecin polygraphe ?

En partant de fonds d’archives laissés par un médecin actif à la fin de l’Ancien Régime, l’objectif de cet article est d’explorer les pistes de recherche et les apports heuristiques d’une analyse de type biographique de ces fonds. Des problématiques quant au statut de la médecine, à l’économie de la pratique, à la relation thérapeutique et au rapport entretenu par le praticien avec le savoir médical seront abordées. Au cours du travail, des questions méthodologiques émergent. Quelle est la spécificité des données issues de la confrontation des données individuelles avec un contexte spécifique ? Est-il possible, à partir du cas singulier, de penser autrement l’histoire du groupe social dont il fait partie ? Quelles autres réalités historiques sont accessibles à partir de l’histoire d’un sujet 

Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells

BACKGROUND: The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs. RESULTS: Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication. CONCLUSIONS: HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known

Characterization of Human Immunodeficiency Virus Type 1 (HIV-1) Diversity and Tropism in 145 Patients With Primary HIV-1 Infection

Viral diversity during primary HIV-1 infection (PHI) relating to transmission mode, HIV-1 subtypes, and viral tropism was revisited. Varying mucosal barriers were not associated with differences in diversities of founder populations. CXCR4-using viruses are present during PHI but remain exceptional case

Characterization of Changes in Serum Anti-Glycan Antibodies in Crohn's Disease – a Longitudinal Analysis

INTRODUCTION: Anti-glycan antibodies are a promising tool for differential diagnosis and disease stratification of patients with Crohn's disease (CD). We longitudinally assessed level and status changes of anti-glycan antibodies over time in individual CD patients as well as determinants of this phenomenon. METHODS: 859 serum samples derived from a cohort of 253 inflammatory bowel disease (IBD) patients (207 CD, 46 ulcerative colitis (UC)) were tested for the presence of anti-laminarin (Anti-L), anti-chitin (Anti-C), anti-chitobioside (ACCA), anti-laminaribioside (ALCA), anti-mannobioside (AMCA) and anti-Saccharomyces cerevisiae (gASCA) antibodies by ELISA. All patients had at least two and up to eleven serum samples taken during the disease course. RESULTS: Median follow-up time for CD was 17.4 months (Interquartile range (IQR) 8.0, 31.6 months) and for UC 10.9 months (IQR 4.9, 21.0 months). In a subgroup of CD subjects marked changes in the overall immune response (quartile sum score) and levels of individual markers were observed over time. The marker status (positive versus negative) remained widely stable. Neither clinical phenotype nor NOD2 genotype was associated with the observed fluctuations. In a longitudinal analysis neither changes in disease activity nor CD behavior led to alterations in the levels of the glycan markers. The ability of the panel to discriminate CD from UC or its association with CD phenotypes remained stable during follow-up. In the serum of UC patients neither significant level nor status changes were observed. CONCLUSIONS: While the levels of anti-glycan antibodies fluctuate in a subgroup of CD patients the antibody status is widely stable over time

Bounce Rock—A shergottite-like basalt encountered at Meridiani Planum, Mars

The Opportunity rover of the Mars Exploration Rover mission encountered an isolated rock fragment with textural, mineralogical, and chemical properties similar to basaltic shergottites. This finding was confirmed by all rover instruments, and a comprehensive study of these results is reported here. Spectra from the miniature thermal emission spectrometer and the Panoramic Camera reveal a pyroxene-rich mineralogy, which is also evident in M&ouml;ssbauer spectra and in normative mineralogy derived from bulk chemistry measured by the alpha particle X-ray spectrometer. The correspondence of Bounce Rock&rsquo;s chemical composition with the composition of certain basaltic shergottites, especially Elephant Moraine (EET) 79001 lithology B and Queen Alexandra Range (QUE) 94201, is very close, with only Cl, Fe, and Ti exhibiting deviations. Chemical analyses further demonstrate characteristics typical of Mars such as the Fe &frasl;Mn ratio and P concentrations. Possible shock features support the idea that Bounce Rock was ejected from an impact crater, most likely in the Meridiani Planum region. Bopolu crater, 19.3 km in diameter, located 75 km to the southwest could be the source crater. To date, no other rocks of this composition have been encountered by any of the rovers on Mars. The finding of Bounce Rock by the Opportunity rover provides further direct evidence for an origin of basaltic shergottite meteorites from Mars.Additional co-authors: Thanasis ECONOMOU, Steven P. GOREVAN, Brian C. HAHN, Göstar KLINGELHÖFER, Timothy J. McCOY, Harry Y. McSWEEN Jr, Douglas W. MING, Richard V. MORRIS, Daniel S. RODIONOV, Steven W. SQUYRES, Heinrich WÄNKE, Shawn P. WRIGHT, Michael B. WYATT, Albert S. YE

Ambiguous Nucleotide Calls From Population-based Sequencing of HIV-1 are a Marker for Viral Diversity and the Age of Infection

The fraction of ambiguous nucleotide calls in bulk sequencing of human immunodeficiency virus type 1 (HIV-1) carries important information on viral diversity and the age of infection. In particular, a fraction of ambiguous nucleotides of >.5% provides evidence against a recent infection event <1 year ag

Impact of baseline SARS-CoV-2 antibody status on syndromic surveillance and the risk of subsequent COVID-19-a prospective multicenter cohort study.

BACKGROUND In a prospective healthcare worker (HCW) cohort, we assessed the risk of SARS-CoV-2 infection according to baseline serostatus. METHODS Baseline serologies were performed among HCW from 23 Swiss healthcare institutions between June and September 2020, before the second COVID-19 wave. Participants answered weekly electronic questionnaires covering information about nasopharyngeal swabs (PCR/rapid antigen tests) and symptoms compatible with coronavirus disease 2019 (COVID-19). Screening of symptomatic staff by nasopharyngeal swabs was routinely performed in participating facilities. We compared numbers of positive nasopharyngeal tests and occurrence of COVID-19 symptoms between HCW with and without anti-nucleocapsid antibodies. RESULTS A total of 4812 HCW participated, wherein 144 (3%) were seropositive at baseline. We analyzed 107,807 questionnaires with a median follow-up of 7.9 months. Median number of answered questionnaires was similar (24 vs. 23 per person, P = 0.83) between those with and without positive baseline serology. Among 2712 HCW with ≥ 1 SARS-CoV-2 test during follow-up, 3/67 (4.5%) seropositive individuals reported a positive result (one of whom asymptomatic), compared to 547/2645 (20.7%) seronegative participants, 12 of whom asymptomatic (risk ratio [RR] 0.22; 95% confidence interval [CI] 0.07 to 0.66). Seropositive HCWs less frequently reported impaired olfaction/taste (6/144, 4.2% vs. 588/4674, 12.6%, RR 0.33, 95% CI 0.15-0.73), chills (19/144, 13.2% vs. 1040/4674, 22.3%, RR 0.59, 95% CI 0.39-0.90), and limb/muscle pain (28/144, 19.4% vs. 1335/4674, 28.6%, RR 0.68 95% CI 0.49-0.95). Impaired olfaction/taste and limb/muscle pain also discriminated best between positive and negative SARS-CoV-2 results. CONCLUSIONS Having SARS-CoV-2 anti-nucleocapsid antibodies provides almost 80% protection against SARS-CoV-2 re-infection for a period of at least 8 months

Risk and symptoms of COVID-19 in health professionals according to baseline immune status and booster vaccination during the Delta and Omicron waves in Switzerland-A multicentre cohort study.

BACKGROUND Knowledge about protection conferred by previous Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and/or vaccination against emerging viral variants allows clinicians, epidemiologists, and health authorities to predict and reduce the future Coronavirus Disease 2019 (COVID-19) burden. We investigated the risk and symptoms of SARS-CoV-2 (re)infection and vaccine breakthrough infection during the Delta and Omicron waves, depending on baseline immune status and subsequent vaccinations. METHODS AND FINDINGS In this prospective, multicentre cohort performed between August 2020 and March 2022, we recruited hospital employees from ten acute/nonacute healthcare networks in Eastern/Northern Switzerland. We determined immune status in September 2021 based on serology and previous SARS-CoV-2 infections/vaccinations: Group N (no immunity); Group V (twice vaccinated, uninfected); Group I (infected, unvaccinated); Group H (hybrid: infected and ≥1 vaccination). Date and symptoms of (re)infections and subsequent (booster) vaccinations were recorded until March 2022. We compared the time to positive SARS-CoV-2 swab and number of symptoms according to immune status, viral variant (i.e., Delta-dominant before December 27, 2021; Omicron-dominant on/after this date), and subsequent vaccinations, adjusting for exposure/behavior variables. Among 2,595 participants (median follow-up 171 days), we observed 764 (29%) (re)infections, thereof 591 during the Omicron period. Compared to group N, the hazard ratio (HR) for (re)infection was 0.33 (95% confidence interval [CI] 0.22 to 0.50, p < 0.001) for V, 0.25 (95% CI 0.11 to 0.57, p = 0.001) for I, and 0.04 (95% CI 0.02 to 0.10, p < 0.001) for H in the Delta period. HRs substantially increased during the Omicron period for all groups; in multivariable analyses, only belonging to group H was associated with protection (adjusted HR [aHR] 0.52, 95% CI 0.35 to 0.77, p = 0.001); booster vaccination was associated with reduction of breakthrough infection risk in groups V (aHR 0.68, 95% CI 0.54 to 0.85, p = 0.001) and H (aHR 0.67, 95% CI 0.45 to 1.00, p = 0.048), largely observed in the early Omicron period. Group H (versus N, risk ratio (RR) 0.80, 95% CI 0.66 to 0.97, p = 0.021) and participants with booster vaccination (versus nonboosted, RR 0.79, 95% CI 0.71 to 0.88, p < 0.001) reported less symptoms during infection. Important limitations are that SARS-CoV-2 swab results were self-reported and that results on viral variants were inferred from the predominating strain circulating in the community at that time, rather than sequencing. CONCLUSIONS Our data suggest that hybrid immunity and booster vaccination are associated with a reduced risk and reduced symptom number of SARS-CoV-2 infection during Delta- and Omicron-dominant periods. For previously noninfected individuals, booster vaccination might reduce the risk of symptomatic Omicron infection, although this benefit seems to wane over time