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HIV prevention with pre-exposure prophylaxis (PrEP) constitutes a major pillar in fighting the ongoing epidemic. While daily oral PrEP adherence may be challenging, long-acting (LA-)PrEP in oral or implant formulations could overcome frequent dosing with convenient administration. The novel drug islatravir (ISL) may be suitable for LA-PrEP, but dose-dependent reductions in CD 4 + T cell and lymphocyte counts were observed at high doses. We developed a mathematical model to predict ISL pro-drug levels in plasma and active intracellular ISL-triphosphate concentrations after oral vs. subcutaneous implant dosing. Using phase II trial data, we simulated antiviral effects and estimated HIV risk reduction for multiple dosages and dosing frequencies. We then established exposure thresholds where no adverse effects on immune cells were observed. Our findings suggest that implants with 56-62 mg ISL offer effective HIV risk reduction without reducing lymphocyte counts. Oral 0.1 mg daily, 3-5 mg weekly, and 10 mg biweekly ISL provide comparable efficacy, but weekly and biweekly doses may affect lymphocyte counts, while daily dosing regimen offered no advantage over existing oral PrEP. Oral 0.5-1 mg on demand provided > 90 % protection, while not being suitable for post-exposure prophylaxis. These findings suggest ISL could be considered for further development as a promising and safe agent for implantable PrEP., (© 2024 The Author(s). CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

RNA dimerization is the noncovalent association of two human immunodeficiency virus-1 (HIV-1) genomes. It is a conserved step in the HIV-1 life cycle and assumed to be a prerequisite for binding to the viral structural protein Pr55 Gag during genome packaging. Here, we developed functional analysis of RNA structure-sequencing (FARS-seq) to comprehensively identify sequences and structures within the HIV-1 5' untranslated region (UTR) that regulate this critical step. Using FARS-seq, we found nucleotides important for dimerization throughout the HIV-1 5' UTR and identified distinct structural conformations in monomeric and dimeric RNA. In the dimeric RNA, key functional domains, such as stem-loop 1 (SL1), polyadenylation signal (polyA) and primer binding site (PBS), folded into independent structural motifs. In the monomeric RNA, SL1 was reconfigured into long- and short-range base pairings with polyA and PBS, respectively. We show that these interactions disrupt genome packaging, and additionally show that the PBS-SL1 interaction unexpectedly couples the PBS with dimerization and Pr55 Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging., (© 2022. The Author(s).)

The combination of the two nucleoside reverse transcriptase inhibitors (NRTI) tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) is used in most highly active antiretroviral therapies for treatment of HIV-1 infection, as well as in pre-exposure prophylaxis against HIV acquisition. Administered as prodrugs, these drugs are taken up by HIV-infected target cells, undergo intracellular phosphorylation and compete with natural deoxynucleoside triphosphates (dNTP) for incorporation into nascent viral DNA during reverse transcription. Once incorporated, they halt reverse transcription. In vitro studies have proposed that TDF and FTC act synergistically within an HIV-infected cell. However, it is unclear whether, and which, direct drug-drug interactions mediate the apparent synergy. The goal of this work was to refine a mechanistic model for the molecular mechanism of action (MMOA) of nucleoside analogues in order to analyse whether putative direct interactions may account for the in vitro observed synergistic effects. Our analysis suggests that depletion of dNTP pools can explain apparent synergy between TDF and FTC in HIV-infected cells at clinically relevant concentrations. Dead-end complex (DEC) formation does not seem to significantly contribute to the synergistic effect. However, in the presence of non-nucleoside reverse transcriptase inhibitors (NNRTIs), its role might be more relevant, as previously reported in experimental in vitro studies., Competing Interests: The authors declare no conflict of interest.

HIV-1 non-B infections have been increasing in Europe for several years. In Germany, subtype A belongs to the most abundant non-B subtypes showing an increasing prevalence of 8.3% among new infections in 2016. Here we trace the origin and examine the current spread of the German HIV-1 subtype A epidemic. Bayesian coalescence and birth-death analyses were performed with 180 German HIV-1 pol sequences and 528 related and publicly available sequences to reconstruct the population dynamics and fluctuations for each of the transmission groups. Our reconstructions indicate two distinct sources of the German subtype A epidemic, with an Eastern European and an Eastern African lineage both cocirculating in the country. A total of 13 German-origin clusters were identified; among these, 6 clusters showed recent activity. Introductions leading to further countrywide spread originated predominantly from Eastern Africa when introduced before 2005. Since 2005, however, spreading introductions have occurred exclusively within the Eastern European clade. Moreover, we observed changes in the main route of subtype A transmission. The beginning of the German epidemic (1985 to 1995) was dominated by heterosexual transmission of the Eastern African lineage. Since 2005, transmissions among German men who have sex with men (MSM) have been increasing and have been associated with the Eastern European lineage. Infections among people who inject drugs dominated between 1998 and 2005. Our findings on HIV-1 subtype A infections provide new insights into the spread of this virus and extend the understanding of the HIV epidemic in Germany. IMPORTANCE HIV-1 subtype A is the second most prevalent subtype worldwide, with a high prevalence in Eastern Africa and Eastern Europe. However, an increase of non-B infections, including subtype A infections, has been observed in Germany and other European countries. There has simultaneously been an increased flow of refugees into Europe and especially into Germany, raising the question of whether the surge in non-B infections resulted from this increased immigration or whether German transmission chains are mainly involved. This study is the first comprehensive subtype A study from a western European country analyzing in detail its phylogenetic origin, the impact of various transmission routes, and its current spread. The results and conclusions presented provide new and substantial insights for virologists, epidemiologists, and the general public health sector. In this regard, they should be useful to those authorities responsible for developing public health intervention strategies to combat the further spread of HIV/AIDS., (Copyright © 2019 American Society for Microbiology.)

Currently, there is no effective vaccine to halt HIV transmission. However, pre-exposure prophylaxis (PrEP) with the drug combination Truvada can substantially decrease HIV transmission in individuals at risk. Despite its benefits, Truvada-based PrEP is expensive and needs to be taken once-daily, which often leads to inadequate adherence and incomplete protection. These deficits may be overcome by next-generation PrEP regimen, including currently investigated long-acting formulations, or patent-expired drugs. However, poor translatability of animal- and ex vivo/in vitro experiments, and the necessity to conduct long-term (several years) human trials involving considerable sample sizes (N>1000 individuals) are major obstacles to rationalize drug-candidate selection. We developed a prophylaxis modelling tool that mechanistically considers the mode-of-action of all available drugs. We used the tool to screen antivirals for their prophylactic utility and identify lower bound effective concentrations that can guide dose selection in PrEP trials. While in vitro measurable drug potency usually guides PrEP trial design, we found that it may over-predict PrEP potency for all drug classes except reverse transcriptase inhibitors. While most drugs displayed graded concentration-prophylaxis profiles, protease inhibitors tended to switch between none- and complete protection. While several treatment-approved drugs could be ruled out as PrEP candidates based on lack-of-prophylactic efficacy, darunavir, efavirenz, nevirapine, etravirine and rilpivirine could more potently prevent infection than existing PrEP regimen (Truvada). Notably, some drugs from this candidate set are patent-expired and currently neglected for PrEP repurposing. A next step is to further trim this candidate set by ruling out compounds with ominous safety profiles, to assess different administration schemes in silico and to test the remaining candidates in human trials., Competing Interests: The authors have declared that no competing interests exist.

To achieve the 90-90-90 goals set by UNAIDS, the number of new HIV infections needs to decrease to approximately 500,000 by 2020. One of the 'five pillars' to achieve this goal is pre-exposure prophylaxis (PrEP). Truvada (emtricitabine-tenofovir) is currently the only medication approved for PrEP. Despite its advantages, Truvada is costly and requires individuals to adhere to the once-daily regimen. To improve PrEP, many next-generation regimen, including long-acting formulations, are currently investigated. However, pre-clinical testing may not guide candidate selection, since it often fails to translate into clinical efficacy. On the other hand, quantifying prophylactic efficacy in the clinic is ethically problematic and requires to conduct long (years) and large (N>1000 individuals) trials, precluding systematic evaluation of candidates and deployment strategies. To prioritize- and help design PrEP regimen, tools are urgently needed that integrate pharmacological-, viral- and host factors determining prophylactic efficacy. Integrating the aforementioned factors, we developed an efficient and exact stochastic simulation approach to predict prophylactic efficacy, as an example for dolutegravir (DTG). Combining the population pharmacokinetics of DTG with the stochastic framework, we predicted that plasma concentrations of 145.18 and 722.23nM prevent 50- and 90% sexual transmissions respectively. We then predicted the reduction in HIV infection when DTG was used in PrEP, PrEP 'on demand' and post-exposure prophylaxis (PEP) before/after virus exposure. Once daily PrEP with 50mg oral DTG prevented 99-100% infections, and 85% of infections when 50% of dosing events were missed. PrEP 'on demand' prevented 79-84% infections and PEP >80% when initiated within 6 hours after virus exposure and continued for as long as possible. While the simulation framework can easily be adapted to other PrEP candidates, our simulations indicated that oral 50mg DTG is non-inferior to Truvada. Moreover, the predicted 90% preventive concentrations can guide release kinetics of currently developed DTG nano-formulations., Competing Interests: The authors have declared that no competing interests exist.

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

Background: Detailed knowledge of the evolutionary potential of polymorphic sites in a viral protein is important for understanding the development of drug resistance in the presence of an inhibitor. We therefore set out to analyse the molecular evolution of the HIV-1 subtype B integrase at the inter-patient level in Germany during a 20-year period prior to the first introduction of integrase strand inhibitors (INSTIs)., Methods: We determined 337 HIV-1 integrase subtype B sequences (amino acids 1-278) from stored plasma samples of antiretroviral treatment-naïve individuals newly diagnosed with HIV-1 between 1986 and 2006. Shannon entropy was calculated to determine the variability at each amino acid position. Time trends in the frequency of amino acid variants were identified by linear regression. Direct coupling analysis was applied to detect covarying sites., Results: Twenty-two time trends in the frequency of amino acid variants demonstrated either single amino acid exchanges or variation in the degree of polymorphy. Covariation was observed for 17 amino acid variants with a temporal trend. Some minor INSTI resistance mutations (T124A, V151I, K156 N, T206S, S230 N) and some INSTI-selected mutations (M50I, L101I, T122I, T124 N, T125A, M154I, G193E, V201I) were identified at overall frequencies >5%. Among these, the frequencies of L101I, T122I, and V201I increased over time, whereas the frequency of M154I decreased. Moreover, L101I, T122I, T124A, T125A, M154I, and V201I covaried with non-resistance-associated variants., Conclusions: Time-trending, covarying polymorphisms indicate that long-term evolutionary changes of the HIV-1 integrase involve defined clusters of possibly structurally or functionally associated sites independent of selective pressure through INSTIs at the inter-patient level. Linkage between polymorphic resistance- and non-resistance-associated sites can impact the selection of INSTI resistance mutations in complex ways. Identification of these sites can help in improving genotypic resistance assays, resistance prediction algorithms, and the development of new integrase inhibitors.

A major aim of Systems Pharmacology is to understand clinically relevant mechanisms of action (MOA) of drugs and to use this knowledge in order to optimize therapy. To enable this mission it is necessary to obtain knowledge on how in vitro testable insights translate into clinical efficacy. Mathematical modeling and data integration are essential components to achieve this goal. Two modeling philosophies are prevalent, each of which in isolation is not sufficient to achieve the above described: In a 'top-down' approach, a minimal pharmacokinetic-pharmacodynamic (PK-PD) model is derived from- and fitted to available clinical data. This model may lack interpretability in terms of mechanisms and may only be predictive for scenarios already covered by the data used to derive it. A 'bottom-up' approach builds on mechanistic insights derived from in vitro/ex vivo experiments, which can be conducted under controlled conditions, but may not be fully representative for the in vivo/clinical situation. In this work, we employ both approaches side-by-side to predict the clinical potency (IC 50 values) of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, emtricitabine and tenofovir. In the 'top-down' approach, this requires to establish the dynamic link between the intracellularly active NRTI-triphosphates (which exert the effect) and plasma prodrug PK and to subsequently link this composite PK model to viral kinetics. The 'bottom-up' approach assesses inhibition of reverse transcriptase-mediated viral DNA polymerization by the intracellular, active NRTI-triphosphates, which has to be brought into the context of target cell infection. By using entirely disparate sets of data to derive and parameterize the respective models, our approach serves as a means to assess the clinical relevance of the 'bottom-up' approach. We obtain very good qualitative and quantitative agreement between 'top-down' vs. 'bottom-up' predicted IC 50 values, arguing for the validity of the 'bottom-up' approach. We noted, however, that the 'top-down' approach is strongly dependent on the sparse and noisy intracellular pharmacokinetic data. All in all, our work provides confidence that we can translate in vitro parameters into measures of clinical efficacy using the 'bottom-up' approach. This may allow to infer the potency of various NRTIs in inhibiting e.g. mutant viruses, to distinguish sources of interaction of NRTI combinations and to assess the efficacy of different NRTIs for repurposing, e.g. for pre-exposure prophylaxis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

While HIV-1 continues to spread, the use of antivirals in preexposure prophylaxis (PrEP) has recently been suggested. Here we present a modular systems pharmacology modeling pipeline, predicting PrEP efficacy of nucleotide reverse transcriptase inhibitors (NRTIs) at the scale of reverse transcription, target-cell, and systemic infection and after repeated viral exposures, akin to clinical trials. We use this pipeline to benchmark the prophylactic efficacy of all currently approved NRTIs in wildtype and mutant viruses. By integrating pharmacokinetic models, we find that intracellular tenofovir-diphosphate builds up too slowly to halt infection when taken "on demand" and that lamivudine may substitute emtricitabine in PrEP combinations. Lastly, we delineate factors confounding clinical PrEP efficacy estimates and provide a method to overcome these. The presented framework is useful to screen and optimize PrEP candidates and strategies and to understand their clinical efficacy by integrating the diverse scales which determine PrEP efficacy., (© 2016 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

An estimated 2.7 million new HIV-1 infections occurred in 2010. `Treatment-for-prevention' may strongly prevent HIV-1 transmission. The basic idea is that immediate treatment initiation rapidly decreases virus burden, which reduces the number of transmittable viruses and thereby the probability of infection. However, HIV inevitably develops drug resistance, which leads to virus rebound and nullifies the effect of `treatment-for-prevention' for the time it remains unrecognized. While timely conducted treatment changes may avert periods of viral rebound, necessary treatment options and diagnostics may be lacking in resource-constrained settings. Within this work, we provide a mathematical platform for comparing different treatment paradigms that can be applied to many medical phenomena. We use this platform to optimize two distinct approaches for the treatment of HIV-1: (i) a diagnostic-guided treatment strategy, based on infrequent and patient-specific diagnostic schedules and (ii) a pro-active strategy that allows treatment adaptation prior to diagnostic ascertainment. Both strategies are compared to current clinical protocols (standard of care and the HPTN052 protocol) in terms of patient health, economic means and reduction in HIV-1 onward transmission exemplarily for South Africa. All therapeutic strategies are assessed using a coarse-grained stochastic model of within-host HIV dynamics and pseudo-codes for solving the respective optimal control problems are provided. Our mathematical model suggests that both optimal strategies (i)-(ii) perform better than the current clinical protocols and no treatment in terms of economic means, life prolongation and reduction of HIV-transmission. The optimal diagnostic-guided strategy suggests rare diagnostics and performs similar to the optimal pro-active strategy. Our results suggest that 'treatment-for-prevention' may be further improved using either of the two analyzed treatment paradigms.

An increase in the proportion of ambiguous base calls in HIV-1 pol population sequences during the course of infection has been demonstrated in different study populations, and sequence ambiguity thresholds to classify infections as recent or nonrecent have been suggested. The aim of our study was to evaluate sequence ambiguities as a candidate biomarker for use in an HIV-1 incidence assay using samples from antiretroviral treatment-naive seroconverters with known durations of infection (German HIV-1 Seroconverter Study). We used 2,203 HIV-1 pol population sequences derived from 1,334 seroconverters to assess the sequence ambiguity method (SAM). We then compared the serological incidence BED capture enzyme immunoassay (BED-CEIA) with the SAM for a subset of 723 samples from 495 seroconverters and evaluated a multianalyte algorithm that includes BED-CEIA results, SAM results, viral loads, and CD4 cell counts for 453 samples from 325 seroconverters. We observed a significant increase in the proportion of sequence ambiguities with the duration of infection. A sequence ambiguity threshold of 0.5% best identified recent infections with 76.7% accuracy. The mean duration of recency was determined to be 208 (95% confidence interval, 196 to 221) days. In the subset analysis, BED-CEIA achieved a significantly higher accuracy than the SAM (84.6 versus 75.5%, P < 0.001) and results were concordant for 64.2% (464/723) of the samples. Also, the multianalyte algorithm did not show better accuracy than the BED-CEIA (83.4 versus 84.3%, P = 0.786). In conclusion, the SAM and the multianalyte algorithm including SAM were inferior to the BED-CEIA, and the proportion of sequence ambiguities is therefore not a preferable biomarker for HIV-1 incidence testing., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance., Methods: Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments., Results: Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates., Conclusion: Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here "in silico" from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of "wet-" and "dry-lab" experimentation may improve our understanding of HIV-1 resistance evolution in the future.

Antiviral pre-exposure prophylaxis (PrEP) through daily drug administration can protect healthy individuals from HIV-1 infection. While PrEP was recently approved by the FDA, the potential long-term consequences of PrEP implementation remain entirely unclear. The aim of this study is to predict the efficacy of different prophylactic strategies with the pro-drug tenofovir-disoproxil-fumarate (TDF) and to assess the sensitivity towards timing- and mode of TDF administration (daily- vs. single dose), adherence and the number of transmitted viruses. We developed a pharmacokinetic model for TDF and its active anabolite tenofovir-diphosphate (TFV-DP) and validated it with data from 4 different trials, including 4 distinct dosing regimes. Pharmacokinetics were coupled to an HIV model and viral decay following TDF mono-therapy was predicted, consistent with available data. Subsequently, a stochastic approach was used to estimate the % infections prevented by (i) daily TDF-based PrEP, (ii) one week TDF started either shortly before, or -after viral exposure and (iii) a single dose oral TDF before viral challenge (sd-PrEP). Analytical solutions were derived to assess the relation between intracellular TFV-DP concentrations and prophylactic efficacy. The predicted efficacy of TDF was limited by a slow accumulation of active compound (TFV-DP) and variable TFV-DP half-life and decreased with increasing numbers of transmitted viruses. Once daily TDF-based PrEP yielded [Formula: see text]80% protection, if at least 40% of pills were taken. Sd-PrEP with 300 mg or 600 mg TDF could prevent [Formula: see text]50% infections, when given at least before virus exposure. The efficacy dropped to [Formula: see text]10%, when given 1 h before 24 h exposure. Efficacy could not be increased with increasing dosage or prolonged administration. Post-exposure prophylaxis poorly prevented infection. The use of drugs that accumulate more rapidly, or local application of tenofovir gel may overcome the need for drug administration long before virus exposure.

Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.

Single-dose nevirapine (sd-NVP) and extended NVP prophylaxis are widely used in resource-constrained settings to prevent vertical HIV-1 transmission. We assessed the pharmacokinetics of sd-NVP in 62 HIV-1-positive pregnant Ugandan woman and their newborns who were receiving sd-NVP prophylaxis to prevent mother-to-child HIV-1 transmission. Based on these data, we developed a mathematical model system to quantify the impact of different sd-NVP regimens at delivery and of extended infant NVP prophylaxis (6, 14, 21, 26, 52, 78, and 102 weeks) on the 2-year risk of HIV-1 transmission and development of drug resistance in mothers and their breast-fed infants. Pharmacokinetic parameter estimates and model-predicted HIV-1 transmission rates were very consistent with other studies. Predicted 2-year HIV-1 transmission risks were 35.8% without prophylaxis, 31.6% for newborn sd-NVP, 19.1% for maternal sd-NVP, and 19.7% for maternal/newborn sd-NVP. Maternal sd-NVP reduced newborn infection predominately by transplacental exchange, providing protective NVP concentrations to the newborn at delivery, rather than by maternal viral load reduction. Drug resistance was frequently selected in HIV-1-positive mothers after maternal sd-NVP. Extended newborn NVP prophylaxis further decreased HIV-1 transmission risks, but an overall decline in cost-effectiveness for increasing durations of newborn prophylaxis was indicated. The total number of infections with resistant virus in newborns was not increased by extended newborn NVP prophylaxis. The developed mathematical modeling framework successfully predicted the risk of HIV-1 transmission and resistance development and can be adapted to other drugs/drug combinations to a priori assess their potential in reducing vertical HIV-1 transmission and resistance spread.