Background:The isolation of many HIV broadly neutralizing antibodies (bnAbs) from people living with HIV (PLWH) and rigorous characterization of their ontogeny has promoted the goal of reverse engineering their natural development as a strategy for achieving an effective preventive HIV vaccine. We previously described the developmental process of CH103, a CD4-binding site (CD4bs)-specific monoclonal antibody, and the associated evolution of HIV Envelopes (Envs) within the person (CH505) from whom it was isolated. A series of monomeric gp120 protein subunit immunogens representing the transmitted founder (TF) and Envs that evolved during infection and optimally reacted with lineage members at each step of the CH103 clone maturation path were evaluated in this placebo controlled randomized vaccine trial to test-- for the first time in humans-- the concept of whether sequential immunization with gp120 monomeric proteins can recapitulate the development of CD4bs B-cell clonal lineages, including CH103. Methods:HIV Vaccine Trials Network 115 (HVTN 115) was a randomized placebo-controlled vaccine trial at US clinical research sites. We tested the safety and immunogenicity of CH505TF gp120 + GLA-SE (Part A), and then the ability of sequential CH505 gp120 proteins (corresponding to CH505's weeks 53 and 78 Envs) + GLA-SE immunizations to induce CD4bs-specific neutralizing antibodies (Part B). We assessed binding and neutralizing antibody responses, antibody dependent cellular cytotoxicity, antibody dependent cellular phagocytosis, T-cell responses and B-cell phenotyping. Results:We enrolled 42 participants between October 2017 and May 2018 for Part A, and 65 participants from December 2020 to October 2022 for Part B. Immunization with the CH505 gp120 proteins adjuvanted with GLA-SE was well tolerated and induced CD4bs-specific B cells and Env-specific plasma antibodies. The plasma neutralizing antibody response was limited to primarily tier 1 autologous and heterologous HIV-1 strains. Blood-derived B-cell repertoire analyses identified CD4bs antibodies that preferentially bound to open-occluded trimeric Envs that exist in an intermediate state between prefusion-closed to CD4-bound open confirmations, consistent with tier 1 HIV neutralizing activity. Conclusions:Together, these results suggest that the low-affinity CH505TF gp120 monomer elicited CD4bs antibodies in the sera and B-cell repertoires of humans. However, our findings also indicate that gp120 monomers are insufficient to induce detectable bnAb precursors to epitopes on native Env trimers. Nonetheless, our data provide a benchmark for comparison with ongoing clinical trials testing high-affinity CH505 Env trimers for induction of CD4bs bnAb precursors.

mRNA technology might accelerate development of an urgently needed preventive human immunodeficiency virus (HIV) vaccine. We evaluated the safety and immunogenicity of three mRNA-encoded envelope trimers, including two doses of soluble and membrane-anchored forms, in a randomized, open-label, phase 1 clinical trial. Vaccines were generally well tolerated, although 6.5% (7 of 108) of participants developed urticaria, a higher proportion than seen with other mRNA vaccines. mRNA-encoded trimers induced strong envelope-specific B and T cell responses. Immunization with membrane-anchored trimers, intended to obscure epitopes at the trimer base targeted by nonneutralizing antibodies, reduced the frequency of base-binding serum antibodies in comparison with soluble trimers. Three immunizations elicited autologous tier 2 serum neutralizing antibodies in 80% of vaccinees receiving the membrane-anchored trimers, in contrast to only 4% receiving the soluble trimer. Thus, with demonstration of more favorable safety, mRNA-encoded membrane-anchored HIV envelope trimers represent a promising platform for HIV vaccine clinical development.

We examined the association between air pollution and neutralizing antibody responses to COVID-19 vaccination in participants enrolled in a phase 3 clinical trial. Seventy-four adults were vaccinated with two doses of the AstraZeneca ChAdOx1 vectored vaccine (AZD1222) (5 x 1010 viral particles) at baseline and day 29, between Aug 28, 2020, to Jan 15, 2021, in Monroe County, NY. SARS-CoV-2 pseudovirus neutralizing ID50 titers (NAb) and total spike protein IgG were assessed at baseline and 15, 29, 43, 57 and 90 days after vaccination. In this pilot study, each participant's dates of neutralization titers were matched to Monroe County ambient concentrations of fine particles (PM2.5; ≤ 2.5 µm), black carbon (BC; marker of traffic), among other particulate and gaseous pollutants. Using linear mixed models, we estimated the association between each interquartile range (IQR) difference in air pollutant concentrations in the 14 days prior to blood collection and antibody responses at each post vaccination timepoint. Though not statistically significant, we observed a 23% reduction in NAb titer (95% CI: -67%, 79%) measured on day 43 (i.e., 14 days after second vaccination) associated with each 0.32 µg/m3 increase in BC concentrations in the prior 14 days. We also observed a 42% increase in spike protein IgG (95% CI: -16%, 141%) measured on day 15 (i.e., 14 days after primary vaccination) associated with each 0.26 µg/m3 increase in BC concentrations in the 14 days prior. A similar pattern for total spike protein IgG was observed at day 29 (42%; 95% CI: -22%, 157%) and 90 (43%; 95% CI: -11%, 127%). Future research will need to explore the possible association between air pollution exposure and antibody response to SARS-CoV-2 vaccination given the potential for compromised vaccine efficacy.

BackgroundSex, race and geographic location may affect vaccine-induced immune responses, yet few preventive HIV vaccine trials have systematically evaluated such effects. The main objective of this study was therefore to examine the role of these factors on vaccine-induced HIV-specific immune responses within the HVTN 204 trial. This randomized, double-blinded, placebo-controlled phase 2 trial enrolled 480 Black and Caucasian adults from Africa and the Americas, who received a trivalent DNA-HIV-1 vaccine prime followed by a rAd5 vector HIV-1 vaccine boost.MethodsAvailable serum samples from baseline and four weeks after the final vaccination boost from Black (n=85, 59% female) and Caucasian (n=49, 51% female) HVTN 204 vaccine recipients from South Africa and the United States of America were studied using an Enzyme-Linked Immunosorbent Assay to determine titers of Envelope-specific IgG1, IgG3 and IgA antibodies. Recognition of linear Envelope peptide-specific IgG responses was mapped in a randomly selected subgroup analysis using a custom-designed peptide microarray (n = 41, 49% female). Associations between vaccine-induced Envelope-specific antibody responses and sex assigned at birth (female or male), race and geographic location were then analyzed by the Mann-Whitney U test, Fisher’s exact test and multivariate logistic regression.ResultsFour weeks post-final vaccination boost, we observed that Envelope-specific antibody titers were significantly increased for IgG1 but reduced for IgA in females (female vs. male median titer: 900 vs. 300, p=0.030 and <100 vs. 100, p=0.007, respectively). Multivariate logistic regression confirmed that female sex increased the odds for higher Envelope-specific IgG1 and low IgA titers compared to males. In terms of antibody epitopes, the V2 region was more frequently recognized in females than males (p=0.008). Race and geographic location had no apparent influence on antibody isotype titers investigated.ConclusionFemale sex was associated with higher vaccine-induced IgG Envelope-specific binding antibody titers and recognition of V2 region of HIV Envelope in HVTN 204 volunteers. No such associations were detected for race or geographic location. Understanding biological factors driving these sex-based differences may improve the design of a new generation of HIV vaccine candidates.

Background: The mRNA platform is under investigation for many vaccines, including HIV-1 vaccines. Objective: To evaluate the safety and tolerability of 3 investigational HIV-1 trimer mRNA vaccines. Design: Safety analysis of mRNA vaccination in a phase 1, randomized, open-label trial. (ClinicalTrials.gov: NCT05217641) Setting: Ten research sites in the United States. Participants: 108 volunteers aged 18 to 55 years without HIV-1. Intervention: Investigational HIV-1 BG505 MD39.3 trimer mRNA vaccines (gp140 soluble trimer, gp151 membrane-bound trimer, and gp151 CD4KO membrane-bound trimer) at doses of 100 and 250 mcg at 0, 2, and 6 months. Measurements: Solicited and unsolicited adverse reactions and events reported during the 12 months after the first vaccination. Results: Participants (n = 108) were randomly assigned to 6 vaccine groups. Mild to moderate local and systemic solicited events were common. Eighty participants reported 190 unsolicited adverse events (AEs); 30 were considered to be related to a study product. Most (73%) related AEs were mild, and the rest were moderate. Among related AEs, urticaria was reported by 7 of 108 participants (7% [95% CI, 3% to 13%]), 4 of whom had unresolved, intermittent urticaria at 12 months. In post hoc analyses, demographic characteristics, history of allergy or medication use, and COVID-19 were not associated with urticaria. In a comparison of participants with versus without urticaria, 100% (7 of 7; CI, 65% to 100%) versus 37% (37 of 101; CI, 28% to 46%) reported previous Moderna COVID-19 vaccination, 29% (2 of 7; CI, 8% to 64%) versus 76% (77 of 101; CI, 67% to 84%) reported previous Pfizer-BioNTech COVID-19 vaccination, and 0% (0 of 7; CI, 0% to 35%) versus 5% (5 of 101; CI, 2% to 11%) reported no previous mRNA COVID-19 vaccination. Limitations: Lack of a placebo group, open-label study, and post hoc evaluation of urticarial risk. Conclusion: Urticarial reactions associated with experimental HIV-1 mRNA vaccines were observed in this trial. Studies to investigate the mechanism and approaches to mitigate these reactions are underway to further advance HIV-1 vaccine research. Primary Funding Source: National Institutes of Health, National Institute of Allergy and Infectious Diseases.

The test-negative design (TND) has been widely used to assess postmarketing COVID-19 vaccine effectiveness but requires further evaluation for this application. To determine whether the TND reliably evaluates vaccine effectiveness against symptomatic COVID-19 using placebo-controlled vaccine efficacy randomized clinical trials (RCTs). This secondary cross-protocol analysis constructed TND study datasets from study sites in 16 countries across 5 continents using the blinded phase cohorts of 5 harmonized phase 3 COVID-19 Prevention Network RCTs: COVE (Coronavirus Vaccine Efficacy and Safety), AZD1222, ENSEMBLE, PREVENT-19 (Prefusion Protein Subunit Vaccine Efficacy Novavax Trial COVID-19), and VAT00008. Participants included adults who received the intended number of doses, experienced COVID-19–like symptoms, and obtained SARS-CoV-2 testing. Start dates ranged from July 27, 2020, to October 19, 2021; data cutoff dates ranged from March 26, 2021, to March 15, 2022. Statistical analysis was performed from May 11, 2023, to February 25, 2025. Participants received vaccines consisting of messenger RNA-1273 (COVE; 2 doses 28 days apart), ChAdOx1 nCoV-19 (AZD1222; 2 doses 28 days apart), Ad26.COV2.S (ENSEMBLE; 1 dose), NVX-CoV2373 (PREVENT-19; 2 doses 21 days apart), CoV2 preS dTM-AS03 (VAT00008; D614) (2 doses 21 days apart), or CoV2 preS dTM-AS03 (D614 plus B.1.351) (VAT00008; 2 doses 21 days apart) or placebo. Main outcomes were symptomatic COVID-19 according to each trial’s primary efficacy definition and the Centers for Disease Control and Prevention definition. Vaccine effectiveness was estimated using targeted maximum likelihood estimation under a semiparametric logistic regression model and ordinary logistic regression. Noncase exchangeability, a core TND assumption for unbiased estimation, was also assessed by estimating vaccine efficacy against non–COVID-19 illness. Among the 12 157 participants included in the analysis, mean (SD) age was 45 (15) years, 6414 were female (53%), 5858 were vaccinated (48%), 2835 experienced primary COVID-19 (23%), and 2992 experienced Centers for Disease Control and Prevention–defined COVID-19 (25%). TND vaccine effectiveness estimates were concordant with RCT vaccine efficacy estimates (concordance correlation coefficient, 0.86 [95% CI, 0.58-0.96] for both outcomes). The semiparametric method had 48% smaller variance estimates than ordinary logistic regression. Noncase exchangeability was generally supported with a median vaccine efficacy against non–COVID-19 illness of 7.7% (IQR, 2.7%-16.8%) across trial cohorts and most 95% CIs including 0. In this cross-protocol analysis, the TND provided reliable inferences on COVID-19 vaccine effectiveness in health care–seeking populations for multiple vaccines and symptom definitions when confounding and selection bias were absent. A machine-learning approach for robust confounding control in postmarketing TND studies was also introduced.

BACKGROUND:Elicitation of broad immune responses is understood to be required for an efficacious preventative HIV vaccine. This Phase 1 randomized controlled trial evaluated whether administration of vaccine antigens separated at multiple injection sites vs combined, fractional delivery at multiple sites affected T-cell breadth compared to standard, single site vaccination.METHODS:We randomized 90 participants to receive recombinant adenovirus 5 (rAd5) vector with HIV inserts gag, pol and env via three different strategies. The Standard group received vaccine at a single anatomic site (n = 30) compared to two polytopic (multisite) vaccination groups: Separated (n = 30), where antigens were separately administered to four anatomical sites, and Fractioned (n = 30), where fractions of each vaccine component were combined and administered at four sites. All groups received the same total dose of vaccine.FINDINGS:CD8 T-cell response rates and magnitudes were significantly higher in the Fractioned group than Standard for several antigen pools tested. CD4 T-cell response magnitudes to Pol were higher in the Separated than Standard group. T-cell epitope mapping demonstrated greatest breadth in the Fractioned group (median 8.0 vs 2.5 for Standard, Wilcoxon p = 0.03; not significant after multiplicity adjustment for co-primary endpoints). IgG binding antibody response rates to Env were higher in the Standard and Fractioned groups vs Separated group.INTERPRETATION:This study shows that the number of anatomic sites for which a vaccine is delivered and distribution of its antigenic components influences immune responses in humans.FUNDING:National Institute of Allergy and Infectious Diseases, NIH.

Key HIV cure strategies involve reversing immune dysfunction and limiting the proliferation of infected T cells. We evaluate the safety of sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, in people with HIV (PWH) and study the impact of sirolimus on HIV-1 reservoir size and HIV-1-specific immunity in a single-arm study of 20 weeks of treatment in PWH on antiretroviral therapy (ART). Sirolimus treatment does not impact HIV-1-specific CD8 T cell responses but leads to a significant decrease in CD4+ T cell-associated HIV-1 DNA levels at 20 weeks of therapy in the primary efficacy population (n = 16; 31% decline, p = 0.008). This decline persists for at least 12 weeks following cessation of the study drug. Sirolimus treatment also leads to a significant reduction in CD4+ T cell cycling and PD-1 expression on CD8+ lymphocytes. These data suggest that homeostatic proliferation of infected cells, an important mechanism for HIV persistence, is an intriguing therapeutic target.

Stabilized trimers preserving the native-like HIV envelope structure may be key components of a preventive HIV vaccine regimen to induce broadly neutralizing antibodies (bnAbs). We evaluated trimeric BG505 SOSIP.664 gp140, formulated with a novel TLR7/8 signaling adjuvant, 3M-052-AF/Alum, for safety, adjuvant dose-finding and immunogenicity in a first-in-healthy adult (n=17), randomized, placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, B-cell and CD4+ T-cell responses emerged post-vaccination. Five vaccinees developed serum autologous tier-2 nAbs (ID50 titer, 1:28-1:8647) after 2-3 doses targeting C3/V5 and/or V1/V2/V3 Env regions by electron microscopy and mutated pseudovirus-based neutralization analyses. Trimer-specific, B-cell-derived monoclonal antibody activities confirmed these results and showed weak heterologous neutralization in the strongest responder. Our findings demonstrate the clinical utility of the 3M-052-AF/alum adjuvant and support further improvements of trimer-based Env immunogens to focus responses on multiple broad nAb epitopes. KEY TAKEAWAY/TAKE-HOME MESSAGES HIV BG505 SOSIP.664 trimer with novel 3M-052-AF/alum adjuvant in humans appears safe and induces serum neutralizing antibodies to matched clade A, tier 2 virus, that map to diverse Env epitopes with relatively high titers. The novel adjuvant may be an important mediator of vaccine response.

Stabilized trimers preserving the native-like HIV envelope structure may be key components of a preventive HIV vaccine regimen to induce broadly neutralizing antibodies (bnAbs). We evaluated trimeric BG505 SOSIP.664 gp140 formulated with a novel TLR7/8 signaling adjuvant, 3M-052-AF/Alum, for safety, adjuvant dose-finding, and immunogenicity in a first-in-healthy adult (n = 17), randomized, and placebo-controlled trial (HVTN 137A). The vaccine regimen appeared safe. Robust, trimer-specific antibody, and B cell and CD4+ T cell responses emerged after vaccination. Five vaccinees developed serum autologous tier 2 nAbs (ID50 titer, 1:28-1:8647) after two to three doses targeting C3/V5 and/or V1/V2/V3 Env regions by electron microscopy and mutated pseudovirus-based neutralization analyses. Trimer-specific, B cell-derived monoclonal antibody activities confirmed these results and showed weak heterologous neutralization in the strongest responder. Our findings demonstrate the clinical utility of the 3M-052-AF/Alum adjuvant and support further improvements of trimer-based Env immunogens to focus responses on multiple broad nAb epitopes.

BACKGROUND. Identifying factors that predict the timing of HIV rebound after treatment interruption will be crucial for designing and evaluating interventions for HIV remission. METHODS. We performed a broad evaluation of viral and immune factors that predict viral rebound (AIDS Clinical Trials Group A5345). Participants initiated antiretroviral therapy (ART) during chronic (N = 33) or early (N = 12) HIV infection with >= 2 years of suppressive ART and restarted ART if they had 2 viral loads >= 1,000 copies/mL after treatment interruption. RESULTS. Compared with chronic-treated participants, early-treated individuals had smaller and fewer transcriptionally active HIV reservoirs. A higher percentage of HIV Gag-specific CD8+ T cell cytotoxic response was associated with lower intact proviral DNA. Predictors of HIV rebound timing differed between early- versus chronic-treated participants, as the strongest reservoir predictor of time to HIV rebound was level of residual viremia in early-treated participants and intact DNA level in chronic-treated individuals. We also identified distinct sets of pre-treatment interruption viral, immune, and inflammatory markers that differentiated participants who had rapid versus slow rebound. CONCLUSION. The results provide an in-depth overview of the complex interplay of viral, immunologic, and inflammatory predictors of viral rebound and demonstrate that the timing of ART initiation modifies the features of rapid and slow viral rebound.

IntroductionThe HVTN 105 vaccine clinical trial tested four combinations of two immunogens - the DNA vaccine DNA-HIV-PT123, and the protein vaccine AIDSVAX B/E. All combinations induced substantial antibody and CD4+ T cell responses in many participants. We have now re-examined the intracellular cytokine staining flow cytometry data using the high-resolution SWIFT clustering algorithm, which is very effective for enumerating rare populations such as antigen-responsive T cells, and also determined correlations between the antibody and T cell responses.MethodsFlow cytometry samples across all the analysis batches were registered using the swiftReg registration tool, which reduces batch variation without compromising biological variation. Registered data were clustered using the SWIFT algorithm, and cluster template competition was used to identify clusters of antigen-responsive T cells and to separate these from constitutive cytokine producing cell clusters.ResultsRegistration strongly reduced batch variation among batches analyzed across several months. This in-depth clustering analysis identified a greater proportion of responders than the original analysis. A subset of antigen-responsive clusters producing IL-21 was identified. The cytokine patterns in each vaccine group were related to the type of vaccine – protein antigens tended to induce more cells producing IL-2 but not IFN-γ, whereas DNA vaccines tended to induce more IL-2+ IFN-γ+ CD4 T cells. Several significant correlations were identified between specific antibody responses and antigen-responsive T cell clusters. The best correlations were not necessarily observed with the strongest antibody or T cell responses.ConclusionIn the complex HVTN105 dataset, alternative analysis methods increased sensitivity of the detection of antigen-specific T cells; increased the number of identified vaccine responders; identified a small IL-21-producing T cell population; and demonstrated significant correlations between specific T cell populations and serum antibody responses. Multiple analysis strategies may be valuable for extracting the most information from large, complex studies.

BACKGROUND:An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. METHODS:HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. FINDINGS:Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. INTERPRETATION:Both vaccine regimens were safe, warranting evaluation in larger trials. FUNDING:US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.

Background. HVTN 120 is a phase 1/2a randomized double-blind placebo-controlled human immunodeficiency virus (HIV) vaccine trial that evaluated the safety and immunogenicity of ALVAC-HIV (vCP2438) and MF59- or AS01(B)-adjuvanted bivalent subtype C gp120 Env protein at 2 dose levels in healthy HIV-uninfected adults.Methods. Participants received ALVAC-HIV (vCP2438) alone or placebo at months 0 and 1. At months 3 and 6, participants received either placebo, ALVAC-HIV (vCP2438) with 200 mu g of bivalent subtype C gp120 adjuvanted with MF59 or AS01(B), or ALVAC-HIV (vCP2438) with 40 mu g of bivalent subtype C gp120 adjuvanted with AS01(B). Primary outcomes were safety and immune responses.Results. We enrolled 160 participants, 55% women, 18-40 years old (median age 24 years) of whom 150 received vaccine and 10 placebo. Vaccines were generally safe and well tolerated. At months 6.5 and 12, CD4(+) T-cell response rates and magnitudes were higher in the AS01(B)-adjuvanted groups than in the MF59-adjuvanted group. At month 12, HIV-specific Env-gp120 binding antibody response magnitudes in the 40 mu g gp120/AS01(B) group were higher than in either of the 200 mu g gp120 groups.Conclusions. The 40 mu g dose gp120/AS01(B) regimen elicited the highest CD4(+) T-cell and binding antibody responses.Clinical Trials Registration. NCT03122223.

ABSTRACT Fosfomycin, approved in the United States only for cystitis, is an attractive alternative for oral treatment of outpatient complicated urinary tract infections (cUTIs) as it has antimicrobial activity against most common uropathogens. The study was a multicenter, randomized, open-label pragmatic superiority clinical trial evaluating the efficacy of oral fosfomycin versus oral levofloxacin strategies in cUTIs (FOCUS study). The trial compared two strategies for initial or step-down oral therapy of cUTI without bacteremia after 0–48 hours of parenteral antibiotic therapy. Subjects were assigned to 3 g of fosfomycin or 750 mg (or dose adjusted for kidney function) of levofloxacin daily for 5–7 days. Clinical and microbiological cures were assessed at the end of therapy (EOT) and test of cure (TOC) (approximately 21 days from the start of antibiotics). The trial did not meet accrual goals; thus, the results were descriptive. Only 51 subjects were included in the microbiological intention-to-treat population. The subjects were mainly females (76%), with a mean age of 46.7 years (standard deviation [SD] = 20.8) and acute pyelonephritis (88%). At the end of therapy, clinical cure remained similar (69% and 68% for fosfomycin and levofloxacin strategies, respectively), and microbiological success was 100% for both strategies. At the test of cure, clinical cure was similar (84% and 86% in the fosfomycin and levofloxacin strategies, respectively); however, a numerically lower microbiological success was observed for fosfomycin (69% compared to 84% for levofloxacin). These limited data suggest that fosfomycin could be an oral alternative as a step-down therapy for the treatment of cUTIs (registry number NCT 03697993). IMPORTANCE Concerns over resistance and safety have been identified in the current treatment regimen for complicated urinary tract infections. Fosfomycin is a drug that is routinely used for the treatment of uncomplicated cystitis. This study shows that fosfomycin could be an oral alternative as step-down therapy for the treatment of complicated urinary tract infections, with a clinical cure rate comparable to levofloxacin but a lower microbiological success rate 3 weeks from start of antibiotics.