Targeting CCRL2 enhances therapeutic outcomes in a tuberculosis mouse model.

Tuberculosis (TB) remains among the leading infectious causes of death. Due to the limited number of antimicrobials in the TB drug discovery pipeline, interest has developed in host-directed approaches to improve TB treatment outcomes. C-C motif chemokine-like receptor 2 (CCRL2) is a unique seven-transmembrane domain receptor that is upregulated by inflammatory signals and mediates leucocyte migration. However, little is known about its role in the setting of TB infection. Here, we show that Mycobacterium tuberculosis (Mtb) infection increases CCRL2 protein expression in macrophages and in mouse lungs. To target selectively CCRL2-expressing cells in vivo, we developed a novel mouse anti-CCRL2 antibody-drug conjugate (ADC) linked with the cytotoxic drug SG3249. We tested its adjunctive therapeutic efficacy against TB when combined with the first-line regimen for drug-susceptible TB (isoniazid, rifampin, pyrazinamide, ethambutol; RHZE). The anti-CCRL2 ADC treatment potentiated RHZE efficacy in Mtb-infected mice and decreased gross lung inflammation. CCRL2 expression in lung dendritic cells and alveolar macrophages was lower in mice receiving anti-CCRL2 ADC treatment + RHZE compared to those receiving RHZE alone or the control group, although the total innate cell populations did not differ across treatment groups. Interestingly, neutrophils were completely absent in the anti-CCRL2 ADC treatment + RHZE group, unlike in the other treatment groups. IFN-γ+ and IL17-Α+ T-cell responses, which are associated with optimal TB control, were also elevated in the anti-CCRL2 ADC treatment + RHZE group. Collectively, our findings suggest that CCRL2-targeting approaches may improve TB treatment outcomes, possibly through selective killing of Mtb-infected innate immune cells.

Therapeutic DNA Vaccine Targeting Mycobacterium tuberculosis Persisters Shortens Curative Tuberculosis Treatment.

Mycobacterium tuberculosis ( Mtb) is one of the leading infectious causes of death worldwide. There is no available licensed therapeutic vaccine that shortens active tuberculosis (TB) disease drug treatment and prevents relapse, despite the World Health Organization's calls. Here, we show that an intranasal DNA vaccine containing a fusion of the stringent response rel Mtb gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3α/CCL20, shortens the duration of curative TB treatment in immunocompetent mice. Compared to the first-line regimen for drug-susceptible TB alone, our novel adjunctive vaccine induced greater Rel Mtb -specific T-cell responses associated with optimal TB control in spleen, blood, lungs, mediastinal lymph nodes, and bronchoalveolar lavage (BAL) fluid. These responses were sustained, if not augmented, over time. It also triggered more effective dendritic cell recruitment, activation, and colocalization with T cells, implying enhanced crosstalk between innate and adaptive immunity. Moreover, it potentiated a 6-month TB drug-resistant regimen, rendering it effective across treatment regimens, and also showed promising results in CD4+ knockout mice, perhaps due to enhanced Rel-specific CD8+ T-cell responses. Notably, our novel fusion vaccine was also immunogenic in nonhuman primates, the gold standard animal model for TB vaccine studies, eliciting antigen-specific T-cell responses in blood and BAL fluid analogous to those observed in protected mice. Our findings have critical implications for therapeutic TB vaccine clinical development in immunocompetent and immunocompromised populations and may serve as a model for defining immunological correlates of therapeutic vaccine-induced protection. One sentence summary: A TB vaccine shortens curative drug treatment in mice by eliciting strong TB-protective immune responses and induces similar responses in macaques.

The heme oxygenase-1 metalloporphyrin inhibitor stannsoporfin enhances the bactericidal activity of a novel regimen for multidrug-resistant tuberculosis in a murine model.

Multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) poses significant challenges to global tuberculosis (TB) control efforts. Host-directed therapies (HDTs) offer a novel approach to TB treatment by enhancing immune-mediated clearance of Mtb. Prior preclinical studies found that the inhibition of heme oxygenase-1 (HO-1), an enzyme involved in heme metabolism, with tin-protoporphyrin IX (SnPP) significantly reduced mouse lung bacillary burden when co-administered with the first-line antitubercular regimen. Here, we evaluated the adjunctive HDT activity of a novel HO-1 inhibitor, stannsoporfin (SnMP), in combination with a novel MDR-TB regimen comprising a next-generation diarylquinoline, TBAJ-876 (S), pretomanid (Pa), and a new oxazolidinone, TBI-223 (O) (collectively, SPaO), in Mtb-infected BALB/c mice. After 4 weeks of treatment, SPaO + SnMP 5mg/kg reduced mean lung bacillary burden by an additional 0.69 log10 (P = 0.01) relative to SPaO alone. As early as 2 weeks post-treatment initiation, SnMP adjunctive therapy differentially altered the expression of pro-inflammatory cytokine genes and CD38, a marker of M1 macrophages. Next, we evaluated the sterilizing potential of SnMP adjunctive therapy in a mouse model of microbiological relapse. After 6 weeks of treatment, SPaO + SnMP 10mg/kg reduced lung bacterial burdens to 0.71 ± 0.23 log10 colony-forming units (CFUs), a 0.78 log-fold greater decrease in lung CFU compared to SpaO alone (P = 0.005). However, adjunctive SnMP did not reduce microbiological relapse rates after 5 or 6 weeks of treatment. SnMP was well tolerated and did not significantly alter gross or histological lung pathology. SnMP is a promising HDT candidate requiring further study in combination with regimens for drug-resistant TB.

The heme oxygenase-1 metalloporphyrin inhibitor stannsoporfin enhances the bactericidal activity of a novel regimen for multidrug-resistant tuberculosis in a murine model.

Multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) poses significant challenges to global tuberculosis (TB) control efforts. Host-directed therapies (HDT) offer a novel approach for TB treatment by enhancing immune-mediated clearance of Mtb. Prior preclinical studies found that inhibition of heme oxygenase-1 (HO-1), an enzyme involved in heme metabolism, with tin-protoporphyrin IX (SnPP) significantly reduced mouse lung bacillary burden when co-administered with the first-line antitubercular regimen. Here we evaluated the adjunctive HDT activity of a novel HO-1 inhibitor, stannsoporfin (SnMP), in combination with a novel MDR-TB regimen comprising a next-generation diarylquinoline, TBAJ-876 (S), pretomanid (Pa), and a new oxazolidinone, TBI-223 (O) (collectively, SPaO) in Mtb-infected BALB/c mice. After 4 weeks of treatment, SPaO + SnMP 5 mg/kg reduced mean lung bacillary burden by an additional 0.69 log10 (P=0.01) relative to SPaO alone. As early as 2 weeks post-treatment initiation, SnMP adjunctive therapy differentially altered the expression of pro-inflammatory cytokine genes, and CD38, a marker of M1 macrophages. Next, we evaluated the sterilizing potential of SnMP adjunctive therapy in a mouse model of microbiological relapse. After 6 weeks of treatment, SPaO + SnMP 10 mg/kg reduced lung bacterial burdens to 0.71 ± 0.23 log10 CFU, a 0.78 log-fold greater decrease in lung CFU compared to SpaO alone (P=0.005). However, adjunctive SnMP did not reduce microbiological relapse rates after 5 or 6 weeks of treatment. SnMP was well tolerated and did not significantly alter gross or histological lung pathology. SnMP is a promising HDT candidate requiring further study in combination with regimens for drug-resistant TB.

An intranasal stringent response vaccine targeting dendritic cells as a novel adjunctive therapy against tuberculosis.

Lengthy tuberculosis (TB) treatment is required to overcome the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine containing a fusion of the relMtb gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3α/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that intramuscular delivery of the MIP-3α/relMtb (fusion) vaccine or intranasal delivery of the relMtb (non-fusion) vaccine potentiate isoniazid activity more than intramuscular delivery of the DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 and 0.5 log10 colony-forming units, respectively; P=0.0002 and P=0.0052), inducing pronounced Mtb-protective immune signatures. The combined approach involving intranasal delivery of the DNA MIP-3α/relMtb fusion vaccine demonstrated the greatest mycobactericidal activity together with isoniazid when compared to each approach alone (absolute reduction of Mtb burden: 1.13 log10, when compared to the intramuscular vaccine targeting relMtb alone; P<0.0001), as well as robust systemic and local Th1 and Th17 responses. This DNA vaccination strategy may be a promising adjunctive approach combined with standard therapy to shorten curative TB treatment, and also serves as proof of concept for treating other chronic bacterial infections.

Sex Differences in Lung Imaging and SARS-CoV-2 Antibody Responses in a COVID-19 Golden Syrian Hamster Model.

In the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males than in females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8 to 10 weeks of age) were inoculated intranasally with 105 50% tissue culture infective dose (TCID50) of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developed more extensive pneumonia as noted on chest computed tomography, and recovered more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including interferon-ß (IFN-ß) and tumor necrosis factor-α (TNF-α), were comparable between the sexes. However, during the recovery phase of infection, females mounted 2-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole-inactivated SARS-CoV-2 and mutant S-RBDs as well as virus-neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2-associated sex differences seen in the human population. IMPORTANCE Men experience more severe outcomes from coronavirus disease 2019 (COVID-19) than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human isolate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in female than in male hamsters. Greater lung pathology during the acute phase combined with lower antiviral antibody responses during the recovery phase of infection in males than in females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection.

Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model.

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 10 5 TCID 50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFNb and TNFa, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population.

Comparison of shortened mosaic HIV-1 vaccine schedules: a randomised, double-blind, placebo-controlled phase 1 trial (IPCAVD010/HPX1002) and a preclinical study in rhesus monkeys (NHP 17-22).

BACKGROUND: Current efficacy studies of a mosaic HIV-1 prophylactic vaccine require four vaccination visits over one year, which is a complex regimen that could prove challenging for vaccine delivery at the community level, both for recipients and clinics. In this study, we evaluated the safety, tolerability, and immunogenicity of shorter, simpler regimens of trivalent Ad26.Mos.HIV expressing mosaic HIV-1 Env/Gag/Pol antigens combined with aluminium phosphate-adjuvanted clade C gp140 protein. METHODS: We did this randomised, double-blind, placebo-controlled phase 1 trial (IPCAVD010/HPX1002) at Beth Israel Deaconess Medical Center in Boston, MA, USA. We included healthy, HIV-uninfected participants (aged 18-50 years) who were considered at low risk for HIV infection and had not received any vaccines in the 14 days before study commencement. We randomly assigned participants via a computer-generated randomisation schedule and interactive web response system to one of three study groups (1:1:1) testing different regimens of trivalent Ad26.Mos.HIV (5 × 1010 viral particles per 0·5 mL) combined with 250 µg adjuvanted clade C gp140 protein. They were then assigned to treatment or placebo subgroups (5:1) within each of the three main groups. Participants and investigators were masked to treatment allocation until the end of the follow-up period. Group 1 received Ad26.Mos.HIV alone at weeks 0 and 12 and Ad26.Mos.HIV plus adjuvanted gp140 at weeks 24 and 48. Group 2 received Ad26.Mos.HIV plus adjuvanted gp140 at weeks 0, 12, and 24. Group 3 received Ad26.Mos.HIV alone at week 0 and Ad26.Mos.HIV plus adjuvanted gp140 at weeks 8 and 24. Participants in the control group received 0·5 mL of 0·9% saline. All study interventions were administered intramuscularly. The primary endpoints were Env-specific binding antibody responses at weeks 28, 52, and 72 and safety and tolerability of the vaccine regimens for 28 days after the injection. All participants who received at least one vaccine dose or placebo were included in the safety analysis; immunogenicity was analysed using the per-protocol population. The IPCAVD010/HPX1002 trial is registered with ClinicalTrials.gov, NCT02685020. We also did a parallel preclinical study in rhesus monkeys to test the protective efficacy of the shortened group 3 regimen. FINDINGS: Between March 7, 2016, and Aug 19, 2016, we randomly assigned 36 participants to receive at least one dose of study vaccine or placebo, ten to each vaccine group and two to the corresponding placebo group. 30 (83%) participants completed the full study, and six (17%) discontinued it prematurely because of loss to follow-up, withdrawal of consent, investigator decision, and an unrelated death from a motor vehicle accident. The two shortened regimens elicited comparable antibody titres against autologous clade C Env at peak immunity to the longer, 12-month regimen: geometric mean titre (GMT) 41 007 (95% CI 17 959-93 636) for group 2 and 49 243 (29 346-82 630) for group 3 at week 28 compared with 44 590 (19 345-102 781) for group 1 at week 52). Antibody responses remained increased (GMT >5000) in groups 2 and 3 at week 52 but were highest in group 1 at week 72. Antibody-dependent cellular phagocytosis, Env-specific IgG3, tier 1A neutralising activity, and broad cellular immune responses were detected in all groups. All vaccine regimens were well tolerated. Mild-to-moderate pain or tenderness at the injection site was the most commonly reported solicited local adverse event, reported by 28 vaccine recipients (93%) and two placebo recipients (33%). Grade 3 solicited systemic adverse events were reported by eight (27%) vaccine recipients and no placebo recipients; the most commonly reported grade 3 systemic symptoms were fatigue, myalgia, and chills. The shortened group 3 regimen induced comparable peak immune responses in 30 rhesus monkeys as in humans and resulted in an 83% (95% CI 38·7-95, p=0·004 log-rank test) reduction in per-exposure acquisition risk after six intrarectal challenges with SHIV-SF162P3 at week 54, more than 6 months after final vaccination. INTERPRETATION: Short, 6-month regimens of a mosaic HIV-1 prophylactic vaccine elicited robust HIV-specific immune responses that were similar to responses elicited by a longer, 12-month schedule. Preclinical data showed partial protective efficacy of one of the short vaccine regimens in rhesus monkeys. Further clinical studies are required to test the suitability of the shortened vaccine regimens in humans. Such shortened regimens would be valuable to increase vaccine delivery at the community level, particularly in resource-limited settings. FUNDING: Ragon Institute (Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University; Cambridge, MA, USA) and Janssen Vaccines & Prevention (Leiden, Netherlands).

Correction: Quantitative approach for the risk assessment of African swine fever and Classical swine fever introduction into the United States through legal imports of pigs and swine products.

[This corrects the article DOI: 10.1371/journal.pone.0182850.].

Quantitative approach for the risk assessment of African swine fever and Classical swine fever introduction into the United States through legal imports of pigs and swine products.

The US livestock safety strongly depends on its capacity to prevent the introduction of Transboundary Animal Diseases (TADs). Therefore, accurate and updated information on the location and origin of those potential TADs risks is essential, so preventive measures as market restrictions can be put on place. The objective of the present study was to evaluate the current risk of African swine fever (ASF) and Classical swine fever (CSF) introduction into the US through the legal importations of live pigs and swine products using a quantitative approach that could be later applied to other risks. Four quantitative stochastic risk assessment models were developed to estimate the monthly probabilities of ASF and CSF release into the US, and the exposure of susceptible populations (domestic and feral swine) to these introductions at state level. The results suggest a low annual probability of either ASF or CSF introduction into the US, by any of the analyzed pathways (5.5*10-3). Being the probability of introduction through legal imports of live pigs (1.8*10-3 for ASF, and 2.5*10-3 for CSF) higher than the risk of legally imported swine products (8.90*10-4 for ASF, and 1.56*10-3 for CSF). This could be caused due to the low probability of exposure associated with this type of commodity (products). The risk of feral pigs accessing to swine products discarded in landfills was slightly higher than the potential exposure of domestic pigs through swill feeding. The identification of the months at highest risk, the origin of the higher risk imports, and the location of the US states most vulnerable to those introductions (Iowa, Minnesota and Wisconsin for live swine and California, Florida and Texas for swine products), is valuable information that would help to design prevention, risk-mitigation and early-detection strategies that would help to minimize the catastrophic consequences of potential ASF/CSF introductions into the US.