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Streptococcus pneumoniae colonizes the nasopharynx and remains a major human pathogen despite antibiotic therapy. Pneumococci cause important diseases including pneumonia, bacteremia, meningitis and otitis media. Many pneumococcal virulence factors contribute to the pathogenesis. On the one hand, capsular polysaccharides, PspA and PspC enable pneumococci to escape host defenses. On the other hand, after the lysis induced by LytA, pneumolysin, teichoic acids, lipoteichoic acids and phosphocholine induce inflammatory reactions which are often deleterious for the host. Others factors as CbpA, neuraminidases, PsaA… participate in adherence, colonization and in the first steps of the infection. A better knowledge of pneumococcal pathogenesis and virulence factors will contribute to the development of new drugs or vaccines. [Copyright &y& Elsevier]

Only one study to date has focused on people living with HIV (PLWH) who refused to participate in a HIV cure/remission-related clinical trial (HCCT)-"decliners" hereafter-that included analytical treatment interruption (ATI). Exploring why these persons refuse may provide valuable information to ensure more ethical recruitment and support in HCCTs within the bigger picture of improving HIV cure research. The qualitative component of the AMEP-EHVA-T02/ANRS-95052 study, called AMEP-Decliners, documented the experiences of French PLWH who refused to participate in EHVA-T02/ANRS-VRI07, a phase II randomized, placebo-controlled HCCT with ATI. AMEP-Decliners comprised semi-structured individual interviews with six decliners in two HIV care sites in France between September 2022 and March 2023. The interviews documented their expectations regarding HCCTs, reasons for refusal, and perceived factors that might have led them to participate. Audio files were transcribed, and an inductive thematic analysis was performed. Surprisingly, the main reason for refusal was not ATI but the trial monitoring. Besides the frequency of appointments, respondents emphasized the incompatibility with their active life. One underlying reason for refusal was that participating would have meant "break[ing] the carefree attitude about the disease," reflecting the substantial psychological burden associated with participation. Finally, respondents perceived that the trial's clinical team did not sufficiently recognize their "normal life" and the level of commitment required to participate, leading them to call for greater involvement by the team: "they have to make an effort too." Results from decliners' discourses highlighted that two levels of commitment to participation must be considered when developing HCCTs: psychological burden and logistical constraints. We suggest allowing home examinations and flexible appointment times, prioritizing face-to-face invitations in order to address the psychological burden associated with HCCT participation, and explaining the reasons for monitoring constraints when they cannot be alleviated. Further studies are necessary to confirm our results.

Background: Current HIV prophylactic vaccines evaluate HIV Env as purified proteins. CD40.HIVRI.Env is an innovative antigen delivery targeting gp140 Env from HIV Clade C 96ZM651 to CD40-expressing antigen-presenting cells, thus harnessing the intrinsic immune-stimulant properties. DNA-HIV-PT123 vaccine encodes 96ZM651 gp140/Gag and 97CN54 Pol/Nef., Methods: Seventy-two HIV-negative volunteers were enrolled between 05/2021 and 10/2022 in a phase 1 placebo-controlled trial conducted in France and Switzerland (N° EudraCT: 2020-001814-40; NCT04842682). Volunteers were randomized (5:1 active versus placebo) in groups receiving either 0.3, 1.0, or 3.0 mg CD40.HIVRI.Env (Hiltonol® adjuvanted) alone or co-administered with DNA-HIV-PT123 at weeks (W) 0, 4, and 24. Safety and immunogenicity were monitored until W48. The primary safety endpoint was the proportion of participants per dose cohort and randomized arm without any grade 3 or 4 biological (abnormal laboratory values), or clinical local or systemic solicited, or unsolicited adverse events between W0 and W48 considered to be related or possibly related to the investigational products., Findings: CD40.HIVRI.Env was well tolerated. Env-specific CD4 + T-cells (IL-2 + or IFN-γ + or TNF + ) were detected in all vaccinees from W6 to W26 and persisted until W48 without a dose-response signal or an effect of DNA-HIV-PT123 co-administration. At W26, IgG response rates (RR) against autologous and nine heterologous gp120/gp140 were 89-100% across all groups and 56-100% at W48. RR against 96ZM651gp70V1V2 were high (90-100%) at W6 and W26 in all groups. Tier1A MW965.26 neutralizing antibody (nAb) titres were detectable in 50-100% of vaccinated individuals at W26, with a dose-response signal, while one volunteer developed nAbs against five Tier2 viruses., Interpretation: CD40.HIVRI.Env alone or administered with DNA-HIV-PT123 was safe and induced early, and sustained anti-Env cellular and V1V2 IgG responses, identified as correlates of protection in the RV144 trial. CD40 targeting Env-based vaccines may be instrumental for inducing protective vaccine responses in prime-boost strategies., Funding: ANRS Emerging infectious diseases (ANRS MIE); Vaccine Research Institute (VRI)., Competing Interests: The authors S.Z., G.Z., and Y.L., are named inventors on patent applications based on this work held by Inserm Transfert. J-D.L. declares having received remuneration by the Global HIV Vaccine Enterprise. O.L. declares receipt of grants, consulting fees and support for attending meetings and/or travel from several vaccine industries. She also participated in Data Safety Monitoring or Advisory Boards of Sanofi and MSS. The remaining authors declare no competing interests. Inserm Transfert provided a license for CD40 targeting vaccines to the biotech company LinKinVax., (© 2024 The Authors.)

Heterologous prime-boost strategies are of interest for HIV vaccine development. The order of prime-boost components could be important for the induction of T cell responses. In this phase I/II multi-arm trial, three vaccine candidates were used as prime or boost: modified vaccinia Ankara (MVA) HIV-B (coding for Gag, Pol, Nef); HIV LIPO-5 (five lipopeptides from Gag, Pol, Nef); DNA GTU-MultiHIV B (coding for Rev, Nef, Tat, Gag, Env gp160 clade B). Healthy human volunteers ( n = 92) were randomized to four groups: 1) MVA at weeks 0/8 + LIPO-5 at weeks 20/28 (M/L); 2) LIPO-5 at weeks 0/8 + MVA at weeks 20/28 (L/M); 3) DNA at weeks 0/4/12 + LIPO-5 at weeks 20/28 (G/L); 4) DNA at weeks 0/4/12 + MVA at weeks 20/28 (G/M). The frequency of IFN-γ-ELISPOT responders at week 30 was 33, 43, 0, and 74%, respectively. Only MVA-receiving groups were further analyzed ( n = 62). Frequency of HIV-specific cytokine-positive (IFN-γ, IL-2, or TNF-α) CD4 + T cells increased significantly from week 0 to week 30 (median change of 0.06, 0.11, and 0.10% for M/L, L/M, and G/M, respectively), mainly after MVA vaccinations, and was sustained until week 52. HIV-specific CD8 + T cell responses increased significantly at week 30 in M/L and G/M (median change of 0.02 and 0.05%). Significant whole-blood gene expression changes were observed 2 wk after the first MVA injection, regardless of its use as prime or boost. An MVA gene signature was identified, including 86 genes mainly related to cell cycle pathways. Three prime-boost strategies led to CD4 + and CD8 + T cell responses and to a whole-blood gene expression signature primarily due to their MVA HIV-B component., (Copyright © 2022 by The American Association of Immunologists, Inc.)

DNA vectors have been widely used as a priming of poxvirus vaccine in prime/boost regimens. Whether the number of DNA impacts qualitatively or quantitatively the immune response is not fully explored. With the aim to reinforce T-cell responses by optimizing the prime-boost regimen, the multicentric EV03/ANRS VAC20 phase I/II trial, randomized 147 HIV-negative volunteers to either 3xDNA plus 1xNYVAC (weeks 0, 4, 8 plus 24; n = 74) or to 2xDNA plus 2xNYVAC (weeks 0, 4 plus 20, 24; n = 73) groups. T-cell responses (IFN-γ ELISPOT) to at least one peptide pool were higher in the 3xDNA than the 2xDNA groups (91% and 80% of vaccinees) (P = 0.049). In the 3xDNA arm, 26 (37%) recipients developed a broader T-cell response (Env plus at least to one of the Gag, Pol, Nef pools) than in the 2xDNA (15; 22%) arms (primary endpoint; P = 0.047) with a higher magnitude against Env (at week 26) (P<0.001). In both groups, vaccine regimens induced HIV-specific polyfunctional CD4 and CD8 T cells and the production of Th1, Th2 and Th17/IL-21 cytokines. Antibody responses were also elicited in up to 81% of vaccines. A higher percentage of IgG responders was noted in the 2xDNA arm compared to the 3xDNA arm, while the 3xDNA group tended to elicit a higher magnitude of IgG3 response against specific Env antigens. We show here that the modulation of the prime strategy, without modifying the route or the dose of administration, or the combination of vectors, may influence the quality of the responses., Competing Interests: The authors have declared that no competing interests exist.

Objectives: This study aimed to determine the timing and level of HIV rebound in blood and seminal plasma and to characterize the HIV rebounding populations after antiretroviral treatment interruption (ATI) in HIV-1-infected participants enrolled in a therapeutic vaccine trial., Design: A 12-week (W) ATI period was proposed at W36 to patients enrolled in the VRI02/ANRS149-LIGHT trial. Paired blood and semen samples were collected before (W32 or W36) and during ATI (W38, W40, W42, W44, and W48)., Methods: HIV-RNA and HIV-DNA were quantified sequentially from blood and semen samples. Ultradeep sequencing (UDS; Roche/454) of partial env HIV-DNA/RNA (C2V3) was performed in both compartments., Results: HIV-RNA rebounded in blood plasma and seminal plasma of all ten participants after ATI [median peak of 5.12 log10 cp/ml (range: 4.61-6.35) and 4.26 log10 cp/ml (3.20-4.67), respectively]. HIV-RNA rebound was detected in blood plasma as soon as W38 in 8/10 patients, and in seminal plasma between W38 and W40 in 8/10 patients. Phylogenetic approaches showed intermingled HIV-RNA populations from plasma and semen during ATI, suggesting a lack of viral compartmentalization between blood and semen., Conclusion: Our data demonstrate rapid and high HIV rebound in semen after ATI, raising concerns about high risk of HIV sexual transmission during HIV cure trials.

Background: Many candidate vaccine strategies against human immunodeficiency virus (HIV) infection are under study, but their clinical development is lengthy and iterative. To accelerate HIV vaccine development optimised trial designs are needed. We propose a randomised multi-arm phase I/II design for early stage development of several vaccine strategies, aiming at rapidly discarding those that are unsafe or non-immunogenic., Methods: We explored early stage designs to evaluate both the safety and the immunogenicity of four heterologous prime-boost HIV vaccine strategies in parallel. One of the vaccines used as a prime and boost in the different strategies (vaccine 1) has yet to be tested in humans, thus requiring a phase I safety evaluation. However, its toxicity risk is considered minimal based on data from similar vaccines. We newly adapted a randomised phase II trial by integrating an early safety decision rule, emulating that of a phase I study. We evaluated the operating characteristics of the proposed design in simulation studies with either a fixed-sample frequentist or a continuous Bayesian safety decision rule and projected timelines for the trial., Results: We propose a randomised four-arm phase I/II design with two independent binary endpoints for safety and immunogenicity. Immunogenicity evaluation at trial end is based on a single-stage Fleming design per arm, comparing the observed proportion of responders in an immunogenicity screening assay to an unacceptably low proportion, without direct comparisons between arms. Randomisation limits heterogeneity in volunteer characteristics between arms. To avoid exposure of additional participants to an unsafe vaccine during the vaccine boost phase, an early safety decision rule is imposed on the arm starting with vaccine 1 injections. In simulations of the design with either decision rule, the risks of erroneous conclusions were controlled <15%. Flexibility in trial conduct is greater with the continuous Bayesian rule. A 12-month gain in timelines is expected by this optimised design. Other existing designs such as bivariate or seamless phase I/II designs did not offer a clear-cut alternative., Conclusions: By combining phase I and phase II evaluations in a multi-arm trial, the proposed optimised design allows for accelerating early stage clinical development of HIV vaccine strategies.

High-dose oral amoxicillin (3 g/day) is the recommended empirical outpatient treatment of community-acquired pneumonia (CAP) in many European guidelines. To investigate the clinical efficacy of this treatment in CAP caused by Streptococcus pneumoniae strains with MICs of amoxicillin > or =2 microg/ml, we used a lethal bacteremic pneumonia model in leukopenic female Swiss mice with induced renal failure to replicate amoxicillin kinetics in humans given 1 g/8 h orally. Amoxicillin (15 mg/kg of body weight/8 h subcutaneously) was given for 3 days. We used four S. pneumoniae strains with differing amoxicillin susceptibility and tolerance profiles. Rapid bacterial killing occurred with an amoxicillin-susceptible nontolerant strain: after 4 h, blood cultures were negative and lung homogenate counts under the 2 log(10) CFU/ml detection threshold (6.5 log(10) CFU/ml in controls, P < 0.01). With an amoxicillin-intermediate nontolerant strain, significant pulmonary bacterial clearance was observed after 24 h (4.3 versus 7.9 log(10) CFU/ml, P < 0.01), and counts were undetectable 12 h after treatment completion. With an amoxicillin-intermediate tolerant strain, 24-h bacterial clearance was similar (5.4 versus 8.3 log(10) CFU/ml, P < 0.05), but 12 h after treatment completion, lung homogenates contained 3.3 log(10) CFU/ml. Similar results were obtained with an amoxicillin-resistant and -tolerant strain. Day 10 survival rates were usually similar across strains. Amoxicillin with pharmacokinetics simulating 1 g/8 h orally in humans is bactericidal in mice with pneumonia due to S. pneumoniae for which MICs were 2 to 4 microg/ml. The killing rate depends not only on resistance but also on tolerance of the S. pneumoniae strains.

The human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic is of unprecedented gravity and is spreading rapidly, notably in the most disadvantaged regions of the world. The search for a preventive vaccine is thus an absolute priority. For over 10 years the French National Agency for AIDS research (ANRS) has been committed to an original program combining basic science and clinical research. The HIV preventive vaccine research program run by the ANRS covers upstream research for the definition of immunogens, animal models, and clinical research to evaluate candidate vaccines. Most researchers in 2004 believe that it should be possible to obtain partial vaccine protection through the induction of a strong and multiepitopic cellular response. Since 1992, the ANRS has set up 15 phases I and II clinical trials in order to evaluate the safety and the capacity of the candidate vaccines for inducing cellular immune responses. The tested candidate vaccines were increasingly complex recombinant canarypox viruses (Alvac) containing sequences coding for certain viral proteins, utilized alone or combined with other immunogens (whole or truncated envelope proteins). ANRS has also been developing an original strategy based on the utilization of lipopeptides. These comprise synthetic fragments of viral proteins associated with lipids that facilitate the induction of a cellular immune response. These approaches promptly allowed the assessment of a prime-boost strategy combining a viral vector and lipopeptides.

Objective: The inherent properties of an invading bacterium may influence the cytokine profile that is ultimately produced. We determined the alterations in proinflammatory (TNF-alpha, IL-1, and IL-6) and anti-inflammatory cytokine (IL-10) expressions in lung tissues within the first 48 h after infection in mice with pneumonia induced by direct intratracheal inoculation of five different pneumococcal strains., Design: Experimental murine model of Streptococcus pneumoniae pneumonia., Subjects: Female BALB/cby mice aged 8-10 weeks., Interventions: Five S. pneumoniae clinical isolates were used in this study. The strains included two serotype 3 strains (P4241 and P30606), two serotype 6 strains (P26772 and P23477), and one serotype 19 strain (P15986). The trachea of anesthetized animals was cannulated via the mouth with a blunt needle, and 50 micro l bacterial suspension of two different inocula (their respective 100% lethal inoculum and the same 10(5) CFU/mouse inoculum of S. pneumoniae strains) were instillated. At predetermined times after pneumococcal infection, i.e., time 0 (preinfection) and 2, 4, 6, 12, 24, and 48 h postinfection in experimental groups, lung tissues were sampled from groups of three mice to quantify lung pro- and anti-inflammatory mediators. The experiments were repeated at least three times., Results: Pneumonia induced by five different pneumococcal isolates resulted in pronounced differences in the local pro- and anti-inflammatory profiles. For example, with a 100% lethal inoculum of S. pneumoniae, the extent and timing of TNF-alpha expression varied greatly among strains, ranging from 2,643 to 10,022 pg/g and from 4 to 48 h, respectively. Moreover, TNF-alpha productions within 48 h postinfection measured by the 48 h area under the curve were differed significantly, ranging from 59,700 to 275,825. These different profiles were not serotype dependent. Comparable results were obtained when IL-1, IL-6, and IL-10 expressions in lung tissues were studied., Conclusions: These findings confirm that the production of the pro- and anti-inflammatory mediators are critically dependent not only upon the different species of bacteria used to establish the experimental infection but also upon the different strains of a specific bacterial species used, i.e., S. pneumoniae in this study. These substantially different host responses were not serotype dependent. Moreover, the profile of lung pro-and anti-inflammatory cytokines within 48 h postinfection, at least in this pneumonia model, was not related to outcome of animals.

In a previous study of a murine peritonitis model, no Streptococcus pneumoniae strains were found that were both clinically penicillin resistant and virulent. This study assessed the relationship between acquired resistance and virulence in single- and double-isogenic penicillin-resistant (Peni-R) mutants obtained by transformation of a virulent penicillin-susceptible recipient strain with pbp2b and pbpX polymerase chain reaction fragments from a Peni-R donor strain. Sequence analysis results of the pbp2b and pbpX alleles from these strains were in keeping with acquired penicillin resistance. The virulence of these strains was significantly reduced, which shows a relationship between beta-lactam resistance and loss of virulence. The phenotype of the 23.2x mutant remained stable after in vivo passage, which suggests that the pbpX gene is involved in growth, whereas virulent revertants of the 23.2b and 23.2b.2x mutants had no change in MIC. Compensatory mutations are implicated in the revival of virulence.

Genetic construction of virulence deficient mutant is a strategy to analyse virulence genes of Streptococcus pneumoniae and was used to virulence factors as capsule, pneumolysin, autolysin and PspA. We perform a model allowing the in vivo positive selection of virulent S. pneumoniae mutants. Mice which are the most susceptible animals to pneumococcal infection, offer the best model for screening virulent S. pneumoniae. Indeed, after intraperitoneal injection of bacterial mix which was composed to a lot of avirulent bacteria (6 log10 CFU per mouse) (V1015 strain, DL50 = 7.05) and few virulent pneumococci (1 to 2 log10 CFU per mouse) (P4241 strain, DL50 < 1), mice cleared all avirulent bacteria but not virulent pneumococci. Thus, mice dead in 3 to 4 days with septicaemia and positive hemoculture contained only virulent strain. This model was validated by in vivo selection of a virulent mutant (V1042, DL50 = 4.1) which was obtained after transformation of avirulent strain V1015 with the genomic fragment of virulent strain P4241. Our model of screening was the only one allowing detection of virulent S. pneumoniae mutants. This new genetic strategy which consisted in gene addition and used mouse as selection agent, could be used to discover new virulence genes required to in vivo bacterial development.

The increasing emergence of penicillin-resistant and multidrug-resistant strains of Streptococcus pneumoniae will create a serious therapeutic problem in coming years. Trovafloxacin is a novel naphthyridone quinolone with promising activity against S. pneumoniae, including penicillin-resistant strains (MIC for 90% of the isolates tested, 0.25 microg/ml). We compared its in vivo efficacy with that of other fluoroquinolones (ciprofloxacin, temafloxacin, and sparfloxacin) and a reference beta-lactam (amoxicillin) in a model of acute experimental pneumonia. Immunocompetent Swiss mice were infected by peroral tracheal delivery of a virulent, penicillin-susceptible strain (MIC, 0.03 microg/ml); leukopenic Swiss mice were infected with three poorly virulent, penicillin-resistant strains (MICs, 4 to 8 microg/ml) and a ciprofloxacin-resistant strain (MIC, 32 microg/ml). Treatments were started 6 h (immunocompetent mice) or 3 h (leukopenic mice) after infection. Doses ranging from 12.5 to 300 mg/kg were given at 12- or 8-h intervals for 3 days. Trovafloxacin (25 mg/kg) was the most effective agent in vivo against penicillin-susceptible and -resistant strains. Corresponding survival rates were 2- to 4-fold higher than with 50-mg/kg sparfloxacin or temafloxacin and 8- to 16-fold higher than with 100-mg/kg ciprofloxacin. The ratios of the area under the concentration-time curve to the MIC in serum and lung tissue were more favorable with trovafloxacin than with the other quinolones. Efficacy in vivo correlated with pharmacokinetic parameters. Trovafloxacin shows potential for the treatment of infections due to penicillin-susceptible and -resistant S. pneumoniae but appears to be ineffective against a ciprofloxacin-resistant strain.