HIV-infected Humans Research Articles

Impacts of Antiretroviral Therapy on the Oral Microbiome and Periodontal Health of Feline Immunodeficiency Virus Positive Cats.

Feline immunodeficiency virus (FIV) is the domestic cat analogue of HIV infection in humans. Both viruses induce oral disease in untreated individuals, with clinical signs that include gingivitis and periodontal lesions. Oral disease manifestations in HIV patients are abated by highly effective combination antiretroviral therapy (cART), though certain oral manifestations persist despite therapy. Microorganisms associated with oral cavity opportunistic infections in patients with HIV cause similar pathologies in cats. To further develop this model, we evaluated characteristics of feline oral health and oral microbiome during experimental FIV infection over an 8-month period following cART. Using 16S metagenomics sequencing, we evaluated gingival bacterial communities at four timepoints in uninfected and FIV-infected cats treated with cART or placebo. Comprehensive oral examinations were also conducted by a veterinary dental specialist over the experimental period. Gingival inflammation was higher in FIV-infected cats treated with placebo compared to cART-treated cats and controls at study endpoint. Oral microbiome alpha diversity increased in all groups, while beta diversity differed among treatment groups, documenting a significant effect of cART therapy on microbiome community composition. This finding has not previously been reported and indicates cART ameliorates immunodeficiency virus-associated oral disease via preservation of oral mucosal microbiota. Further, this study illustrates the value of the FIV animal model for investigations of mechanistic associations and therapeutic interventions for HIV oral manifestations.

A remarkable genetic shift in a transmitted/founder virus broadens antibody responses against HIV-1

A productive HIV-1 infection in humans is often established by transmission and propagation of a single transmitted/founder (T/F) virus, which then evolves into a complex mixture of variants during the lifetime of infection. An effective HIV-1 vaccine should elicit broad immune responses in order to block the entry of diverse T/F viruses. Currently, no such vaccine exists. An in-depth study of escape variants emerging under host immune pressure during very early stages of infection might provide insights into such a HIV-1 vaccine design. Here, in a rare longitudinal study involving HIV-1 infected individuals just days after infection in the absence of antiretroviral therapy, we discovered a remarkable genetic shift that resulted in near complete disappearance of the original T/F virus and appearance of a variant with H173Y mutation in the variable V2 domain of the HIV-1 envelope protein. This coincided with the disappearance of the first wave of strictly H173-specific antibodies and emergence of a second wave of Y173-specific antibodies with increased breadth. Structural analyses indicated conformational dynamism of the envelope protein which likely allowed selection of escape variants with a conformational switch in the V2 domain from an α-helix (H173) to a β-strand (Y173) and induction of broadly reactive antibody responses. This differential breadth due to a single mutational change was also recapitulated in a mouse model. Rationally designed combinatorial libraries containing 54 conformational variants of V2 domain around position 173 further demonstrated increased breadth of antibody responses elicited to diverse HIV-1 envelope proteins. These results offer new insights into designing broadly effective HIV-1 vaccines.

A remarkable genetic shift in a transmitted/founder virus broadens antibody responses against HIV-1.

A productive HIV-1 infection in humans is often established by transmission and propagation of a single transmitted/founder (T/F) virus, which then evolves into a complex mixture of variants during the lifetime of infection. An effective HIV-1 vaccine should elicit broad immune responses in order to block the entry of diverse T/F viruses. Currently, no such vaccine exists. An in-depth study of escape variants emerging under host immune pressure during very early stages of infection might provide insights into such a HIV-1 vaccine design. Here, in a rare longitudinal study involving HIV-1 infected individuals just days after infection in the absence of antiretroviral therapy, we discovered a remarkable genetic shift that resulted in near complete disappearance of the original T/F virus and appearance of a variant with H173Y mutation in the variable V2 domain of the HIV-1 envelope protein. This coincided with the disappearance of the first wave of strictly H173-specific antibodies and emergence of a second wave of Y173-specific antibodies with increased breadth. Structural analyses indicated conformational dynamism of the envelope protein which likely allowed selection of escape variants with a conformational switch in the V2 domain from an α-helix (H173) to a β-strand (Y173) and induction of broadly reactive antibody responses. This differential breadth due to a single mutational change was also recapitulated in a mouse model. Rationally designed combinatorial libraries containing 54 conformational variants of V2 domain around position 173 further demonstrated increased breadth of antibody responses elicited to diverse HIV-1 envelope proteins. These results offer new insights into designing broadly effective HIV-1 vaccines.

Toll-like Receptor 2 Mediated Immune Regulation in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Toll-like receptors (TLRs) are crucial to the innate immune response. They regulate inflammatory reactions by initiating the production of pro-inflammatory cytokines and chemokines. TLRs also play a role in shaping the adaptive immune responses. While this protective response is important for eliminating infectious pathogens, persistent activation of TLRs may result in chronic immune activation, leading to detrimental effects. The role of TLR2 in regulating HIV-1 infection in vivo has yet to be well described. In this study, we used an SIV-infected rhesus macaque model to simulate HIV infection in humans. We evaluated the plasma of the macaques longitudinally and found a significant increase in the soluble TLR2 (sTLR2) level after SIV infection. We also observed an increase in membrane-bound TLR2 (mb-TLR2) in cytotoxic T cells, B cells, and NK cells in PBMC and NK cells in the gut after infection. Our results suggest that sTLR2 regulates the production of various cytokines and chemokines, including IL-18, IL-1RA, IL-15, IL-13, IL-9, TPO, FLT3L, and IL-17F, as well as chemokines, including IP-10, MCP-1, MCP-2, ENA-78, GRO-α, I-TAC, Fractalkine, SDF-1α, and MIP-3α. Interestingly, these cytokines and chemokines were also upregulated after the infection. The positive correlation between SIV copy number and sTLR2 in the plasma indicated the involvement of TLR2 in the regulation of viral replication. These cytokines and chemokines could directly or indirectly regulate viral replication through the TLR2 signaling pathways. When we stimulated PBMC with the TLR2 agonist in vitro, we observed a direct induction of various cytokines and chemokines. Some of these cytokines and chemokines, such as IL-1RA, IL-9, IL-15, GRO-α, and ENA-78, were positively correlated with sTLR2 in vivo, highlighting the direct involvement of TLR2 in the regulation of the production of these factors. Our findings suggest that TLR2 expression may be a target for developing new therapeutic strategies to combat HIV infection.

Very low prevalence of Mansonella perstans-specific cell-free DNA in serum samples of Ghanaian HIV patients.

Mansonellosis is a widely neglected helminth disease which is predominantly observed in tropical regions. This study was conducted to assess potential associations of the prevalence of circulating Mansonella perstans-specific cell-free DNA in human serum and HIV infection in Ghanaian individuals. For this purpose, serum samples obtained from Ghanaian HIV-patients (n = 989) and non-HIV-infected Ghanaian control individuals (n = 91) were subjected to real-time PCR targeting the ITS-(internal transcribed spacer-)2 sequence of M. perstans and Mansonella sp. Deux. Mansonella-specific cell-free DNA was detected in serum samples of only 2 HIV-positive and 0 HIV-negative individuals, making any reliable conclusions on potential associations between HIV and mansonellosis in tropical Ghana unfeasible. Future epidemiological studies on hypothetical associations between mansonellosis and HIV infections should focus more specifically on high-endemicity settings for both Mansonella spp.-infections and HIV-infections, include higher case numbers and be based on real-time PCR from whole blood rather than from serum, in which only circulating parasite DNA but no more cell-bound parasite DNA can be detected. However, the study did not show associations of HIV infections in Ghanaian individuals with Mansonella worm loads high enough to detect cell-free Mansonella DNA in serum by PCR.

Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction, and cellular dNTP levels.

We introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of sterile α motif and HD domain-containing protein 1 (SAMHD1) T592 phosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiologically relevant context. A proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses and their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.

Extended safety and tolerability of subcutaneous CAP256V2LS and VRC07-523LS in HIV-negative women: study protocol for the randomised, placebo-controlled double-blinded, phase 2 CAPRISA 012C trial

IntroductionWomen-controlled HIV prevention technologies that overcome adherence challenges of available daily oral pre-exposure prophylaxis and give women a choice of options are urgently needed. Broadly neutralising monoclonal antibodies (bnAbs) administered...

Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection.

CD4+ T follicular helper (TFH) cells are key targets for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication and contribute to the virus reservoir under antiretroviral therapy (ART). Here, we describe a novel CD3+ CD20+ double-positive (DP) lymphocyte subset, resident in secondary lymphoid organs of humans and rhesus macaques (RMs), that appear predominantly after membrane exchange between TFH and B cells. DP lymphocytes are enriched in cells displaying a TFH phenotype (CD4+ PD1hi CXCR5hi), function (interleukin 21 positive [IL-21+]), and gene expression profile. Importantly, expression of CD40L upon brief in vitro mitogen stimulation identifies, by specific gene-expression signatures, DP cells of TFH-cell origin versus those of B-cell origin. Analysis of 56 RMs showed that DP cells (i) significantly increase following SIV infection, (ii) are reduced after 12 months of ART in comparison to pre-ART levels, and (iii) expand to a significantly higher frequency following ART interruption. Quantification of total SIV-gag DNA on sorted DP cells from chronically infected RMs showed that these cells are susceptible to SIV infection. These data reinforce earlier observations that CD20+ T cells are infected and expanded by HIV infection, while suggesting that these cells phenotypically overlap activated CD4+ TFH cells that acquire CD20 expression via trogocytosis and can be targeted as part of therapeutic strategies aimed at HIV remission. IMPORTANCE The HIV reservoir is largely composed of latently infected memory CD4+ T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4+ T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART. In lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, we show that CD3+ CD20+ lymphocytes emerge after membrane exchange between T cells and B cells and are enriched in phenotypic, functional, and gene expression profiles found in T follicular helper cells. Furthermore, in SIV-infected rhesus macaques, these cells expand following experimental infection and after interruption of ART and harbor SIV DNA at levels similar to those found in CD4+ T cells; thus, CD3+ CD20+ lymphocytes are susceptible to SIV infection and can contribute to SIV persistence.

The effects of Avemar treatment on feline immunodeficiency virus infected cell cultures.

In addition to standard highly active antiretroviral therapy protocols, complementary therapies using natural compounds are widely used by human immunodeficiency virus (HIV)-infected human patients. One such compound is the fermented wheat germ extract (FWGE), named Avemar. In this study, we investigate the effects of Avemar in a feline-acquired immunodeficiency syndrome model. MBM lymphoid cells were acutely infected by the American feline immunodeficiency virus (FIV)-Petaluma (FIV-Pet) and the European FIV Pisa-M2 strains. FL-4 lymphoid cells, continuously producing FIV-Pet, served as a model for chronic infection. Crandell Rees feline kidney (CRFK) cells were infected by either FIV-Pet or feline adenovirus (FeAdV) as a model for transactivation and opportunistic viral infection. Cell cultures were treated pre- and post-infection with serial dilutions of spray-dried FWGE (Avemar pulvis, AP), a standardized active ingredient in commercial Avemar products. Residual FIV and FeAdV infectivity was quantified. In a concentration-dependent manner, AP inhibited replication of FIV strains in MBM and CRFK cells by 3-5log. Low AP concentration prevented FIV-Pet release from FL-4 cells. Higher concentrations destroyed virus-producing cells with cytopathic effects resembling apoptosis. AP strongly inhibited FeAdV production inside CRFK cells but not in HeLa cells. Adenovirus particles are then released via the disintegration of CRFK cells. This report is the first to describe the antiviral effects of Avemar. Further studies are required to confirm its in vitro and in vivo effects and to investigate the potential for its use as a nutraceutical in FIV-infected felines or HIV-infected humans. Avemar, as a single nutraceutical, inhibits FIV replication and destroys retrovirus carrier cells. An important conclusion is that prolonged Avemar treatment might reduce the number of retrovirus-producing cells in the host.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

Factors Associated with Utilization of Sexual and Reproductive Health Services among the Youth in Lira City West, Northern Uganda: A Cross-Sectional Study.

The youth in Uganda, as in much of sub-Saharan Africa, face numerous sexual and reproductive health (SRH) challenges, from human immunodeficiency virus HIV infection, and unsafe abortions to unwanted pregnancies. This study, therefore, assessed the utilization of sexual and reproductive health services and associated factors among the youth in Lira city west, northern Uganda. This was a cross-sectional study conducted in January 2023 among 386 young people (15-24 years) in Lira city west division, Lira city. Multistage cluster sampling technique was used to recruit our study participants. Data were collected using an interviewer-administered questionnaire. Data were analyzed using SPSS version 23, descriptive statistics, cross-tabulation, bivariate, and multivariate logistic regression analyses. All variables were set by p values less than 0.05 and reported adjusted odds ratio with its 95% confidence interval. The utilization of SRH services was 42.0% (162/386) among the study participants. Family planning, voluntary HIV counseling and testing (VCT), and general counseling services were the most utilized SRH services in the past 12 months. Young people who were aware of SRH services (AOR: 0.24; 95% CI: 0.08-0.74), were aware of a reproductive health facility (AOR, 4.24; 95% CI: 1.62-11.09), discussed SRH issues with peers/friends (AOR, 3.98; 95% CI: 1.53-10.33), had a sexual partner (AOR, 10.00; 95% CI: 4.05-24.69), had sexual intercourse (AOR, 4.59; 95% CI: 2.18-9.69), and had access to SRH services (AOR, 2.68; 95% CI: 1.12-6.40) were more likely to utilize SRH services compared to their counterparts. This study showed a low utilization of sexual and reproductive health services among youth in Lira city west, northern Uganda. Being aware of SRH services, awareness of reproductive health facilities, discussing SRH issues with peers, having sexual intercourse, having a sexual partner, and access to SRH services were independently associated with the utilization of SRH services. Therefore, there is a need to strengthen sustainable multisector approaches aimed at improving awareness and access to sexual and reproductive health services among the youth.

Visceral leishmaniasis and HIV coinfection in Brazil: epidemiological profile and spatial patterns.

Geographical overlap of human visceral leishmaniasis (HVL) and HIV infection favors occurrences of HVL/HIV coinfection. The increasing numbers of cases of HVL/HIV coinfection are a worldwide concern and knowledge of the factors involved can help in developing preventive measures. We aimed to identify spatial patterns and describe the epidemiological profile of HVL/HIV coinfection in Brazil from 2007 to 2015. This was an ecological study, in which thematic maps were created through geoprocessing tools, based on secondary data obtained from open-access platforms, to identify priority areas for interventions for controlling HLV/HIV coinfection, using the TerraView 4.2.2 software. We found spatial autocorrelations between the HVL/HIV rates of neighboring municipalities according to the global Moran index (0.10; p<0.01). The highest HVL/HIV rate was in the central-western region. Among the epidemiological characteristics according to the regions, an increasing trend in the annual variation rate was observed, with positive values over the years and statistical significance (p<0.05) in the North with 1.62 (95% CI 0.57 to 2.69; p=0.02) and Northeast with 6.41 (95% CI 2.60 to 10.37; p=0.006). Similarly, increasing trends were observed in the states of Maranhão with 21.34 (95% CI 13.99 to 9.16; p<0.001) and Sergipe with 5.44 (95% CI 0.61 to 10.50; p=0.04). Our results showed spatial overlap between occurrences of HVL and HIV with spatial patterns of HVL/HIV coinfection, thus revealing that the numbers of cases reported are increasing. Identifying areas with higher coinfection indices contributes to applying interventions and control measures among targeted populations, to prevent new cases.

Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus

SIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated intravenous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.

Animal models for studies of HIV-1 brain reservoirs.

The HIV-1 often evades a robust antiretroviral-mediated immune response, leading to persistent infection within anatomically privileged sites including the CNS. Continuous low-level infection occurs in the presence of effective antiretroviral therapy (ART) in CD4+ T cells and mononuclear phagocytes (MP; monocytes, macrophages, microglia, and dendritic cells). Within the CNS, productive viral infection is found exclusively in microglia and meningeal, perivascular, and choroidal macrophages. MPs serve as the principal viral CNS reservoir. Animal models have been developed to recapitulate natural human HIV-1 infection. These include nonhuman primates, humanized mice, EcoHIV, and transgenic rodent models. These models have been used to study disease pathobiology, antiretroviral and immune modulatory agents, viral reservoirs, and eradication strategies. However, each of these models are limited to specific component(s) of human disease. Indeed, HIV-1 species specificity must drive therapeutic and cure studies. These have been studied in several model systems reflective of latent infections, specifically in MP (myeloid, monocyte, macrophages, microglia, and histiocyte cell) populations. Therefore, additional small animal models that allow productive viral replication to enable viral carriage into the brain and the virus-susceptible MPs are needed. To this end, this review serves to outline animal models currently available to study myeloid brain reservoirs and highlight areas that are lacking and require future research to more effectively study disease-specific events that could be useful for viral eradication studies both in and outside the CNS.

HIV Reservoirs: Modeling, Quantification, and Approaches to a Cure.

Biomedical research in animal models depends heavily on nonhuman primates (NHP) (Phillips et al., Am J Primatol 76(9):801-827, 2014). In their physiology, neurobiology, and, most importantly, their susceptibility to infectious diseases and subsequent immune responses, NHPs have many parallels with humans (Rhesus Macaque Genome Sequencing and Analysis Consortium et al., Science 316(5822):222-234, 2007). Different species of NHPs have served as important animal models for numerous infectious diseases spanning a wide range of pathogens (Gardner and Luciw, ILAR J 49(2):220-255, 2008). As a result of recognizing their utility in HIV research, NHPs have contributed to groundbreaking studies of disease pathogenesis, vaccination, and curative research (London et al., Lancet 2(8355):869-873, 1983; Henrickson et al., Lancet 1 (8321):388-390, 1983). Many African NHPs are considered natural hosts for SIV in which SIV infection is usually nonprogressive and does not cause acquired immunodeficiency syndrome (AIDS) (Chahroudi et al., Science 335(6073):1188-1193, 2012; Taaffe et al., J Virol 84(11):5476-5484, 2010). However, cross-species transmission of SIV strains to other NHPs or to humans (nonnatural hosts) leads to progressive disease and AIDS (Paiardini et al., Annu Rev Med 60:485-495, 2009). In particular, SIV infection of Asian rhesus macaques recapitulates many features of HIV infection in humans and therefore has become a widely used approach for contemporary HIV research into virus persistence and cure strategies (Gardner and Luciw, FASEB J 3(14):2593-2606, 1989). There are multiple factors that should be considered in HIV/SIV studies using NHPs including the particular monkey species and geographic background, age and sex, certain genetic properties, virus strain, route and dose of infection, interventional treatments, and prespecified study outcomes. Here, we discuss consideration of these factors to address specific questions in HIV cure research.

Circulating integrin α4 β7+ CD4 T cells are enriched for proliferative transcriptional programs in HIV infection.

HIV preferentially infects α4 β7+ CD4 T cells, forming latent reservoirs that contribute to HIV persistence during antiretroviral therapy. However, the properties of α4 β7+ CD4 T cells in blood and mucosal compartments remain understudied. Employing two distinct models of HIV infection, HIV-infected humans and simian-human immunodeficiency virus (SHIV)-infected rhesus macaques, we show that α4 β7+ CD4 T cells in blood are enriched for genes regulating cell cycle progression and cellular metabolism. Unlike their circulating counterparts, rectal α4 β7+ CD4 T cells exhibited a core tissue-residency gene expression program. These features were conserved across primate species, indicating that the environment influences memory T-cell transcriptional networks. Our findings provide an important molecular foundation for understanding the role of α4 β7 in HIV infection.

Broadly Neutralizing Antibodies for HIV-1 Prevention.

Given the absence of an effective vaccine for protection against HIV-1 infection, passive immunization strategies that utilize potent broadly neutralizing antibodies (bnAbs) to block acquisition of HIV-1 are being rigorously pursued in the clinical setting. bnAbs have demonstrated robust protection in preclinical animal models, and several leading bnAb candidates have shown favorable safety and pharmacokinetic profiles when tested individually or in combinations in early phase human clinical trials. Furthermore, passive administration of bnAbs in HIV-1 infected individuals has resulted in prolonged suppression of viral rebound following interruption of combination antiretroviral therapy, and robust antiviral activity when administered to viremic individuals. Recent results from the first efficacy trials testing repeated intravenous administrations of the anti-CD4 binding site bnAb VRC01 have demonstrated positive proof of concept that bnAb passive immunization can confer protection against HIV-1 infection in humans, but have also highlighted the considerable barriers that remain for such strategies to effectively contribute to control of the epidemic. In this review, we discuss the current status of clinical studies evaluating bnAbs for HIV-1 prevention, highlight lessons learned from the recent Antibody Mediated Prevention (AMP) efficacy trials, and provide an overview of strategies being employed to improve the breadth, potency, and durability of antiviral protection.

Monkeying around with MAIT Cells: Studying the Role of MAIT Cells in SIV and Mtb Co-Infection.

There were an estimated 10 million new cases of tuberculosis (TB) disease in 2019. While over 90% of individuals successfully control Mycobacterium tuberculosis (Mtb) infection, which causes TB disease, HIV co-infection often leads to active TB disease. Despite the co-endemic nature of HIV and TB, knowledge of the immune mechanisms contributing to the loss of control of Mtb replication during HIV infection is lacking. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that target and destroy bacterially-infected cells and may contribute to the control of Mtb infection. Studies examining MAIT cells in human Mtb infection are commonly performed using peripheral blood samples. However, because Mtb infection occurs primarily in lung tissue and lung-associated lymph nodes, these studies may not be fully translatable to the tissues. Additionally, studies longitudinally examining MAIT cell dynamics during HIV/Mtb co-infection are rare, and lung and lymph node tissue samples from HIV+ patients are typically unavailable. Nonhuman primates (NHP) provide a model system to characterize MAIT cell activity during Mtb infection, both in Simian Immunodeficiency Virus (SIV)-infected and SIV-naïve animals. Using NHPs allows for a more comprehensive understanding of tissue-based MAIT cell dynamics during infection with both pathogens. NHP SIV and Mtb infection is similar to human HIV and Mtb infection, and MAIT cells are phenotypically similar in humans and NHPs. Here, we discuss current knowledge surrounding MAIT cells in SIV and Mtb infection, how SIV infection impairs MAIT cell function during Mtb co-infection, and knowledge gaps to address.

Ultrasensitive detection and quantification of viral nucleic acids with Raindance droplet digital PCR (ddPCR).

Sensitive detection of viral nucleic acids is critically important for diagnosis and monitoring of the progression of infectious diseases such as those caused by SARS-CoV2, HIV-1, and other viruses. In HIV-1 infection cases, assessing the efficacy of treatment interventions that are superimposed on combination antiretroviral therapy (cART) has benefited tremendously from the development of sensitive HIV-1 DNA and RNA quantitation assays. Simian immunodeficiency virus (SIV) infection of Rhesus macaques is similar in many key aspects to human HIV-1 infection and consequently this non-human primate (NHP) model has and continues to prove instrumental in evaluating HIV prevention, treatment and eradication approaches. Cell and tissue associated HIV-1 viral nucleic acids have been found to serve as useful predictors of disease outcome and indicators of treatment efficacy, highlighting the value of and the need for sensitive detection of viruses in cells/tissues from infected individuals or animal models. However, viral nucleic acid detection and quantitation in such sample sources can often be complicated by high nucleic acid input (that is required to detect ultralow level viruses in, for example, cure research) or inhibitors, leading to reduced detection sensitivity and under-quantification, and confounded result interpretation. Here, we present a step-by-step procedure to quantitatively recover cell/tissue associated viral DNA and RNA, using SIV-infected Rhesus macaque cells and tissues as model systems, and subsequently quantify the viral DNA and RNA with an ultrasensitive SIV droplet digital PCR (ddPCR) assay and reverse transcription ddPCR (RT-ddPCR) assay, respectively, on the Raindance ddPCR platform. The procedure can be readily adapted for a broad range of applications where highly sensitive nucleic acid detection and quantitation are required.

In pursuit of sensitivity: Lessons learned from viral nucleic acid detection and quantification on the Raindance ddPCR platform.

Sensitive PCR detection of viral nucleic acids plays a critical role in infectious disease research, diagnosis and monitoring. In the context of SARS-CoV-2 detection, recent reports indicate that digital PCR-based tests are significantly more sensitive than traditional qPCR tests. Numerous factors can influence digital PCR reaction sensitivity. In this review, using a model for human HIV infection and the Raindance ddPCR platform as an example, we describe technical aspects that contribute to sensitive viral signal detection in DNA and RNA from tissue samples, which often harbor viral reservoirs and serve as better predictors of disease outcome and indicators of treatment efficacy.