Maternal and fetal safety outcomes after in utero stem cell injection: A systematic review

Objective: To investigate the maternal and fetal safety of In utero stem cell transplantation (IUSCT). Methods: Medline®, Embase and Cochrane library (1967−2023) search for publications reporting IUSCT in humans. Two reviewers independently screened abstracts and full-text papers. Results: Sixty six transplantation procedures in 52 fetuses were performed for haemoglobinopathies (n = 14), red cell/bleeding disorders (n = 4), immunodeficiencies (n = 15), storage disorders (n = 7), osteogenesis imperfecta (n = 2) and healthy fetuses (n = 10). The average gestational age was 18.9 weeks; of procedures reporting the injection route, cells were delivered by intraperitoneal (n = 37), intravenous (n = 19), or intracardiac (n = 4) injection or a combination (n = 3); most fetuses received one injection (n = 41). Haematopoietic (n = 40) or mesenchymal (n = 12) stem cells were delivered. The cell dose was inconsistently reported (range 1.8−3.3 × 109 cells total (n = 27); 2.7−5.0 × 109/kg estimated fetal weight (n = 17)). The acute fetal procedural complication rate was 4.5% (3/66); the acute fetal mortality rate was 3.0% (2/66). Neonatal survival was 69.2% (36/52). Immediate maternal and pregnancy outcomes were reported in only 30.8% (16/52) and 44.2% (23/52) of cases respectively. Four fetal/pregnancy outcomes would also classify as ≥ Grade 2 maternal adverse events. Conclusions: Short-, medium-, and long-term maternal and fetal adverse events should be reported in all IUSCT studies

Exposure to Apoptotic Activated CD4+ T Cells Induces Maturation and APOBEC3G- Mediated Inhibition of HIV-1 Infection in Dendritic Cells

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4+ T cells (ApoInf) or apoptotic uninfected activated CD4+ T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4+ T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection

Generation of neutralizing antibodies and divergence of SIVmac239 in cynomolgus macaques following short-term early antiretroviral therapy.

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4(+) T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 10(4) copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4(+) T-cell numbers, low viral load and limited viral divergence

Methods for detection of HIV-2/SIV infections

The main objective of this thesis was to develop and improve laboratory methods for detection of HIV-2 in infected humans and detection of HIV-2/SIV in experimentally infected cynomolgus monkeys. Early in the HIV epidemic no assays were commercially available for detection of HIV-2/SIV infections and laboratory testing often relied on cross-reactivity in the assays. Indirect, serological methods are commonly used to demonstrate HIV infection. The screening for anti-HIV antibodies by ELISA is usually followed by confirmation of positive screening results by immunoblot. The sensitivity of different immunoblots for confirmation of HIV-2 infection was evaluated and the presence and form of the envelope proteins in the different WB assays were investigated. One of three commercial WB kits tested had a lower sensitivity for detection of HIV-2 seroconversion, due to lack of gp125, when applying WHO criteria for interpretation of results. There was no difference in sensitivity for detection of HIV-2 seroconversion dependent on the form of the transmembranous glycoprotein. An HIV-2 PCR assay was optimized with regard to primers, other reagents and sample preparation method. The assay was used together with an HIV-1 PCR assay to evaluate the accuracy of antibody assays designed to discriminate between HIV- 1 and HIV-2. We found a high correlation between dual serological reactivity and dual HIV-1 and HIV-2 infection as demonstrated by PCR. An antigen capture ELISA for direct detection of HIV-2/SIV antigen was developed and a PCR assay to discriminate between HIV-2 and SIV was established. These methods were applied to monitoring infection in monkeys experimentally infected with HIV-2 and SIV. We were able to demonstrate protection against infection or disease progression in HIV-2 preinfected cynomolgus monkeys challenged either intravenously or intrarectally with pathogenic SIV. By applying a method for quantification of plasma viral load, we demonstrated that a viral RNA threshold value can be used to predict disease progression not only in infected monkeys but also in monkeys immunized with live HIV-2 SBL-6669 or with MVA expressing HIV-2 env, gag-pol and challenged with pathogenic SIV. A plasma virus load greater than 105 RNA equivalents/ml of plasma at 6 to 12 weeks after inoculation was predictive of a rapid disease progression while less than 104 RNA equivalents/ml of plasma at 6 to 12 weeks was indicative of slow progression to AIDS. The tests presented in this thesis are necessary tools in our research regarding HIV-2 infections in humans and HIV-2/SIV vaccine studies in monkeys. Both the antigen test and the WB assay are less expensive than the current commercially available tests. The discriminatory PCR assays are crucial for investigation of dual HIV- 1 and HIV-2 infection in humans as well as for vaccine research involving coinfection with HIV-2 and SIV in monkeys. Furthermore, the establishment of a threshold SIV RNA value predictive of disease outcome in the vaccinated monkeys allows shorter follow-up periods in the vaccine experiments. The main objective of this thesis was to develop and improve laboratory methods for detection of HIV-2 in infected humans and detection of HIV-2/SIV in experimentally infected cynomolgus monkeys. Early in the HIV epidemic no assays were commercially available for detection of HIV-2/SIV infections and laboratory testing often relied on cross-reactivity in the assays. Indirect, serological methods are commonly used to demonstrate HIV infection. The screening for anti-HIV antibodies by ELISA is usually followed by confirmation of positive screening results by immunoblot. The sensitivity of different immunoblots for confirmation of HIV-2 infection was evaluated and the presence and form of the envelope proteins in the different WB assays were investigated. One of three commercial WB kits tested had a lower sensitivity for detection of HIV-2 seroconversion, due to lack of gp125, when applying WHO criteria for interpretation of results. There was no difference in sensitivity for detection of HIV-2 seroconversion dependent on the form of the transmembranous glycoprotein. An HIV-2 PCR assay was optimized with regard to primers, other reagents and sample preparation method. The assay was used together with an HIV-1 PCR assay to evaluate the accuracy of antibody assays designed to discriminate between HIV- 1 and HIV-2. We found a high correlation between dual serological reactivity and dual HIV-1 and HIV-2 infection as demonstrated by PCR. An antigen capture ELISA for direct detection of HIV-2/SIV antigen was developed and a PCR assay to discriminate between HIV-2 and SIV was established. These methods were applied to monitoring infection in monkeys experimentally infected with HIV-2 and SIV. We were able to demonstrate protection against infection or disease progression in HIV-2 preinfected cynomolgus monkeys challenged either intravenously or intrarectally with pathogenic SIV. By applying a method for quantification of plasma viral load, we demonstrated that a viral RNA threshold value can be used to predict disease progression not only in infected monkeys but also in monkeys immunized with live HIV-2 SBL-6669 or with MVA expressing HIV-2 env, gag-pol and challenged with pathogenic SIV. A plasma virus load greater than 105 RNA equivalents/ml of plasma at 6 to 12 weeks after inoculation was predictive of a rapid disease progression while less than 104 RNA equivalents/ml of plasma at 6 to 12 weeks was indicative of slow progression to AIDS. The tests presented in this thesis are necessary tools in our research regarding HIV-2 infections in humans and HIV-2/SIV vaccine studies in monkeys. Both the antigen test and the WB assay are less expensive than the current commercially available tests. The discriminatory PCR assays are crucial for investigation of dual HIV- 1 and HIV-2 infection in humans as well as for vaccine research involving coinfection with HIV-2 and SIV in monkeys. Furthermore, the establishment of a threshold SIV RNA value predictive of disease outcome in the vaccinated monkeys allows shorter follow-up periods in the vaccine experiments

Generation of neutralizing antibodies and divergence of SIVmac239 in cynomolgus macaques following short-term early antiretroviral therapy

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4+ T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 104 copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4+ T-cell numbers, low viral load and limited viral divergence