Genomic evolution of human respiratory syncytial virus during a decade (2013-2023): bridging the path to monoclonal antibody surveillance.

OBJECTIVES: This study investigated the prevalence, genetic diversity, and evolution of human respiratory syncytial virus (HRSV) in Barcelona from 2013 to 2023. METHODS: Respiratory specimens from patients with RTI suspicion at Hospital Universitari Vall d'Hebron were collected from October 2013 to May 2023 for laboratory-confirmation of respiratory viruses. Next-generation sequencing was performed in randomly-selected samples with Illumina technology. Phylogenetic analyses of whole genome sequences were performed with BEAST v1.10.4. Signals of selection and evolutionary pressures were inferred by population dynamics and evolutionary analyses. Mutations in major surface proteins were genetic and structurally characterised, emphasizing those within antigenic epitopes. RESULTS: Analyzing 139,625 samples, 5.3% were HRSV-positive (3008 HRSV-A, 3882 HRSV-B, 56 HRSV-A and -B, and 495 unsubtyped HRSV), with a higher prevalence observed in the paediatric population. Pandemic-related shifts in seasonal patterns returned to normal in 2022-2023. A total of 198 whole-genome sequences were obtained for HRSV-A (6.6% of the HRSV-A positive samples) belonging to GA2.3.5 lineage. For HRSV-B, 167 samples were sequenced (4.3% of the HRSV-B positive samples), belonging to GB5.0.2, GB5.0.4a and GB5.0.5a. HRSV-B exhibited a higher evolution rate. Post-SARS-CoV-2 pandemic, both subtypes showed increased evolutionary rates and decreased effective population size initially, followed by a sharp increase. Analyses indicated negative selective pressure on HRSV. Mutations in antigenic epitopes, including S276N and M274I in palivizumab-targeted site II, and I206M, Q209R, and S211N in nirsevimab-targeted site Ø, were identified. DISCUSSION: Particularly in the context of the large-scale use in 2023-2024 season of nirsevimab, continuous epidemiological and genomic surveillance is crucial.

The emergence, impact, and evolution of human metapneumovirus variants from 2014 to 2021 in Spain.

BACKGROUND: Human metapneumovirus (HMPV) is an important aetiologic agent of respiratory tract infection (RTI). This study aimed to describe the prevalence, genetic diversity, and evolutionary dynamics of HMPV. METHODS: Laboratory-confirmed HMPV were characterised based on partial-coding G gene sequences with MEGA.v6.0. WGS was performed with Illumina, and evolutionary analyses with Datamonkey and Nextstrain. RESULTS: HMPV prevalence was 2.5%, peaking in February-April and with an alternation in the predominance of HMPV-A and -B until the emergence of SARS-CoV-2, not circulating until summer and autumn-winter 2021, with a higher prevalence and with the almost only circulation of A2c111dup. G and SH proteins were the most variable, and 70% of F protein was under negative selection. Mutation rate of HMPV genome was 6.95 × 10-4 substitutions/site/year. CONCLUSION: HMPV showed a significant morbidity until the emergence of SARS-CoV-2 pandemic in 2020, not circulating again until summer and autumn 2021, with a higher prevalence and with almost the only circulation of A2c111dup, probably due to a more efficient immune evasion mechanism. The F protein showed a very conserved nature, supporting the need for steric shielding. The tMRCA showed a recent emergence of the A2c variants carrying duplications, supporting the importance of virological surveillance.

Chimeric Human Papillomavirus-16 Virus-like Particles Presenting HIV-1 P18I10 Peptide: Expression, Purification, Bio-Physical Properties and Immunogenicity in BALB/c Mice.

Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG (p = 0.6409) was observed as Gardasil anti-HPV vaccine-immunized mice. Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1.

Detection of reassortant influenza B strains from 2004 to 2015 seasons in Barcelona (Catalonia, Spain) by whole genome sequencing.

BACKGROUND: Influenza B viruses (FLUBV) have segmented genomes which enables the virus to evolve by segment reassortment. Since the divergence of both FLUBV lineages, B/Victoria/2/87 (FLUBV/VIC) and B/Yamagata/16/88 (FLUBV/YAM), PB2, PB1 and HA have kept the same ancestor, while some reassortment events in the other segments have been reported worldwide. The aim of the present study was to find out reassortment episodes in FLUBV strains detected in cases attended at Hospital Universitari Vall d'Hebron and Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) from 2004 to 2015 seasons. METHODS: From October 2004 to May 2015, respiratory specimens were received from patients with respiratory tract infection suspicion. Influenza detection was carried out by either cell culture isolation, immunofluorescence or PCR-based assays. A RT-PCR was performed to distinguish both lineages by agarose gel electrophoresis. Whole genome amplification was performed using the universal primer set by Zhou et al. in 2012, and subsequently sequenced using Roche 454 GS Junior platform. Bioinformatic analysis was performed to characterise the sequences with B/Malaysia/2506/2007 and B/Florida/4/2006 corresponding sequences as reference of (B/VIC) and (B/YAM), respectively. RESULTS: A total of 118 FLUBV (75 FLUBV/VIC and 43 FLUBV/YAM), from 2004 to 2006, 2008-2011 and 2012-2015 seasons, were studied. The whole genome of 58 FLUBV/VIC and 42 FLUBV/YAM viruses was successfully amplified. Based on HA sequences, most FLUBV/VIC viruses (37; 64%) belonged to clade 1A (B/Brisbane/60/2008) except to 11 (19%), which fell within clade 1B (B/HongKong/514/2009) and 10 (17%) to B/Malaysia/2506/2004. Nine (20%) FLUBV/YAM viruses belonged to clade 2 (B/Massachusetts/02/2012), 18 (42%) to clade 3 (B/Phuket/3073/2013) and 15 (38%) fell within Florida/4/2006. Numerous intra-lineage reassortments in PB2, PB1, NA and NS were found in 2 2010-2011 viruses. An important inter-lineage reassortment event from 2008 to 2009 (11), 2010-2011 (26) and 2012-2013 (3) FLUBV/VIC (clade 1) strains to FLUBV/YAM (clade 3) was found, in addition to 1 reassortant NS in 2010-2011 B/VIC virus. CONCLUSIONS: Intra- and inter-lineage reassortment episodes were revealed by WGS. While PB2-PB1-HA remained in complex, NP and NS reassortant viruses were found in both lineages. Despite reassorment events are not often, the characterisation only by HA and NA sequences might be underestimating their detection.

Patients with drug-abuse poisoning with and without HIV infection: differential characteristics. / Intoxicaciones por drogas de abuso: características diferenciales en población VIH.

OBJECTIVES: Persons with HIV infection who use illicit drugs have higher morbidity and mortality rates than nonusers with or without HIV infection. The objetive were to detect differences between acute poisoning from illicit drugs in patients with and without HIV infection who are attended in hospital emergency departments, and to identify independent factors associated with a worse prognosis, defined by hospital admission or death. MATERIAL AND METHODS: Observational study in 2 hospitals between January 2017 and 31 December 2021. Included were patients with acute illicit drug poisoning with and without HIV infection. RESULTS: Information for 1132 patients was included. The mean (SD) ages of patients with and without HIV infection, respectively, were 38.9 (9.6) years and 32.6 (10.4) years. In patients with HIV, the main drugs used were opioids (279 [85.3%]), cocaine (226 [69.1%]), and amphetamines (153 [46.8%]. None in this group were on methadone substitution therapy for opioid addiction. In patients without HIV infection the main drugs were cocaine (372 [47.2%]) and cannabis (238 [33.8%]). Alcohol was used along with illicit drugs in 387 cases. Multivariate analysis showed that the only variables independently associated with a poor prognosis were HIV infection (odds ratio [OR], 2.19 [1.29-3.11], P .003), age (OR, 1.20 [1.01-1.05], P .003), and acute poisoning from benzodiazepines (OR, 3.48 [2.14-5.66], P .001). The area under the receiver operating characteristic curve of the model was 0.717. CONCLUSION: Certain characteristics distinguish the illicit drug use of patients with HIV infection. HIV infection, age, and the use of benzodiazepines are independently associated with a poor prognosis in acute poisonings.

OBJETIVO: La población VIH, consumidora de drogas de abuso (DA), tiene mayor morbimortalidad en relación con los no consumidores y no VIH. Se investiga si existen diferencias en las intoxicaciones agudas (IA) por DA, en pacientes VIH y no VIH atendidos en los servicios de urgencias hospitalarios (SUH), y se identifican factores independientes de mal pronóstico, definido por ingreso o fallecimiento. METODO: Estudio bicéntrico y observacional de 1 de enero de 2017 al 31 de diciembre de 2021. Se incluyeron pacientes VIH y no VIH atendido en dos SUH por intoxicación por DA. Se recogieron variables demográficas y la sustancia consumida. La variable de resultado principal fue mal pronóstico, definido como ingreso o muerte a los 30 días. RESULTADOS: Se recogieron 1.132 pacientes. La edad media de los pacientes VIH fue 39 ± 10 años, y 33 ± 10 años para los no VIH. En la población VIH predominaron los opiáceos 279 (85,3%) (ninguno de ellos estaba en tratamiento sustitutivo con metadona), la cocaína 226 (30,9%) y las anfetaminas 153 (69,1%), mientras que en la no VIH predominaron la cocaína 372 (47,2%) y el cannabis 238 (33,8%). El etanol se asoció con otras DA en 387 pacientes. El análisis multivariado mostró que las únicas variables independientes de mal pronóstico fueron el VIH [OR 2,19 (1,29-3,11), p 0,003], la edad [OR 1,20 (1,01-1,05), p 0,003], y la IA por benzodiacepinas (BDZ) [OR 3,48 (2,14-5,66), p 0,001], con un área bajo la curva de la característica operativa del receptor de este modelo de 0,717. CONCLUSIONES: Existen diferencias en las características de las IA en pacientes VIH. La infección VIH, la edad y el consumo de BZD son factores independientes de mal pronóstico en las IA.

Molecular characterization and clinical impact of human bocavirus at a tertiary hospital in Barcelona (Catalonia, Spain) during the 2014-2017 seasons.

PURPOSE: The aim was to describe the prevalence, molecular epidemiology and clinical manifestations of human bocavirus (HBoV) in patients attended at a tertiary hospital in Barcelona, Spain. METHODS: From October 2014 to May 2017, respiratory specimens from paediatric patients were collected for respiratory viruses' laboratory-confirmation. Phylogenetic analyses from partial VP1 sequences were performed from all HBoV laboratory-confirmed specimens. Clinical features were retrospectively studied. RESULTS: 178/10271 cases were HBoV laboratory-confirmed. The median age was 1.53 (IQR 1.0-2.3). Co-detection was highly reported (136; 76%). All viruses belonged into HBoV1 genotype but one into HBoV2. Non-reported mutations were observed and two sites were suggestive to be under negative selection. 61% (109/178) cases had lower RTI (LRTI), of whom 84 had co-detections (77%) and 76 had comorbidities (70%). LRTI was the cause of hospitalization in 85 out of 109 cases (78%), and no differences were found regarding severity factors during hospitalization between co- and single-detections, except for median length of respiratory support, which was longer in cases with co-detections. CONCLUSIONS: Close monitoring of predominant HBoV1 showed a high similarity between viruses. The presence of comorbidities might explain the high prevalence of LRTI. Symptomatology in HBoV single-detected cases suggest that HBoV is a true pathogen.

Emergence of Delta and Omicron variants carrying resistance-associated mutations in immunocompromised patients undergoing sotrovimab treatment with long-term viral excretion.

OBJECTIVES: To monitor the early emergence of genetic mutations related to reduced susceptibility to monoclonal anti-body (mAb)-based treatment in immunocompromised patients with long-term viral excretion using whole-genome sequencing at a tertiary university hospital in Barcelona, Spain. METHODS: Serial severe acute respiratory syndrome coronavirus 2-positive samples (mid-December 2021-mid-March 2022) from eight immunosuppressed, fully vaccinated patients (for solid-organ transplantation or haematologic malignancies) with long-term viral excretion despite undergoing mAb therapy (sotrovimab) for coronavirus disease 2019 were selected. Whole-genome sequencing was performed following the ARTIC, version 4.1, protocol on the MiSeq platform. Mutations in the coding sequence of the spike protein with a frequency of ≥5% were studied. RESULTS: A total of 37 samples from the studied cases were analysed. All the cases, except one, were confirmed to have the Omicron variant BA.1; one had Delta (AY.100). Thirty-four different mutations were detected within the receptor-binding domain of the spike protein in 62.5% of patients, eight of which were not lineage related and located in the sotrovimab target epitope (P337L, E340D, E340R, E340K, E340V, E340Q, R346T and K356T). Except for P337L, all changes showed a significant increase in frequency or fixation after the administration of sotrovimab. Some of them have been associated with either reduced susceptibility to mAb therapy, such as those at position 340, or the acquisition of a new glycosylation site (346 and 356 positions). CONCLUSIONS: This study highlights the importance of monitoring for early in vivo selection of mutations associated with reduced susceptibility to mAb therapy, especially in immunocompromised patients receiving anti-viral drugs, whose immune response is not able to control viral replication, resulting in long-term viral shedding, and those receiving selective evolution pressure. Virologic surveillance of genetically resistant viruses to available anti-viral therapies is considered a priority for both patients and the community.

The frequency of defective genomes in Omicron differs from that of the Alpha, Beta and Delta variants.

The SARS-CoV-2 Omicron variant emerged showing higher transmissibility and possibly higher resistance to current COVID-19 vaccines than other variants dominating the global pandemic. In March 2020 we performed a study in clinical samples, where we found that a portion of genomes in the SARS-CoV-2 viral population accumulated deletions immediately before the S1/S2 cleavage site (furin-like cleavage site, PRRAR/S) of the spike gene, generating a frameshift and appearance of a premature stop codon. The main aim of this study was to determine the frequency of defective deletions in prevalent variants from the first to sixth pandemic waves in our setting and discuss whether the differences observed might support epidemiological proposals. The complete SARS-CoV-2 spike gene was deeply studied by next-generation sequencing using the MiSeq platform. More than 90 million reads were obtained from respiratory swab specimens of 78 COVID-19 patients with mild infection caused by the predominant variants circulating in the Barcelona city area during the six pandemic waves: B.1.5, B.1.1, B.1.177, Alpha, Beta, Delta, and Omicron. The frequency of defective genomes found in variants dominating the first and second waves was similar to that seen in Omicron, but differed from the frequencies seen in the Alpha, Beta and Delta variants. The changing pattern of mutations seen in the various SARS-CoV-2 variants driving the pandemic waves over time can affect viral transmission and immune escape. Here we discuss the putative biological effects of defective deletions naturally occurring before the S1/S2 cleavage site during adaption of the virus to human infection.

Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014-2021.

To determine molecular epidemiology and clinical features of enterovirus D68 (EV-D68) infections, we reviewed EV-D68-associated respiratory cases at a hospital in Barcelona, Spain, during 2014-2021. Respiratory samples were collected from hospitalized patients or outpatients with symptoms of acute respiratory tract infection or suggestive of enterovirus infection. Enterovirus detection was performed by real-time multiplex reverse transcription PCR and characterization by phylogenetic analysis of the partial viral protein 1 coding region sequences. From 184 patients with EV-D68 infection, circulating subclades were B3 (80%), D1 (17%), B2 (1%), and A (<1%); clade proportions shifted over time. EV-D68 was detected mostly in children (86%) and biennially (2016, 2018, 2021). In patients <16 years of age, the most common sign/symptom was lower respiratory tract infection, for which 11.8% required pediatric intensive care unit admission and 2.3% required invasive mechanical ventilation; neurologic complications developed in 1. The potential neurotropism indicates that enterovirus surveillance should be mandatory.

A year living with SARS-CoV-2: an epidemiological overview of viral lineage circulation by whole-genome sequencing in Barcelona city (Catalonia, Spain).

Herein, we describe the genetic diversity of circulating SARS-CoV-2 viruses by whole-genome sequencing (WGS) in Barcelona city (Catalonia, Spain) throughout the first four pandemic waves. From weeks 11/2020-24/2021, SARS-CoV-2-positive respiratory samples were randomly selected per clinical setting (80% from primary care or 20% from the hospital), age group, and week. WGS was performed following the ARTICv3 protocol on MiSeq or NextSeq2000 Illumina platforms. Nearly complete consensus sequences were used for genetic characterization based on GISAID and PANGOLIN nomenclatures. From 2475 samples, 2166 (87%) were fully sequenced (78% from primary care and 22% from hospital settings). Multiple genetic lineages were co-circulating, but four were predominant at different periods. While B.1.5 (50.68%) and B.1.1 (32.88%) were the major lineages during the first pandemic wave, B.1.177 (66.85%) and B.1.1.7 (83.80%) were predominant during the second, third, and fourth waves, respectively. Almost all (96.4%) were carrying D614G mutation in the S protein, with additional mutations that define lineages or variants. But some mutations of concern, such as E484K from B.1.351 and P.1 lineages are currently under monitoring, together with those observed in the receptor-binding domain or N-terminal domain, such as L452R and T478K from B.1.617.2 lineage. The fact that a predominant lineage was observed in each pandemic wave suggests advantageous properties over other contemporary co-circulating variants. This genetic variability should be monitored, especially when a massive vaccination campaign is ongoing because the potential selection and emergence of novel antigenic SARS-CoV-2 strains related to immunological escapement events.

Chimeric Human Papillomavirus-16 Virus-like Particles Presenting P18I10 and T20 Peptides from HIV-1 Envelope Induce HPV16 and HIV-1-Specific Humoral and T Cell-Mediated Immunity in BALB/c Mice.

In this study, the HIV-1 P18I10 CTL peptide derived from the V3 loop of HIV-1 gp120 and the T20 anti-fusion peptide of HIV-1 gp41 were inserted into the HPV16 L1 capsid protein to construct chimeric HPV:HIV (L1:P18I10 and L1:T20) VLPs by using the mammalian cell expression system. The HPV:HIV VLPs were purified by chromatography. We demonstrated that the insertion of P18I10 or T20 peptides into the DE loop of HPV16 L1 capsid proteins did not affect in vitro stability, self-assembly and morphology of chimeric HPV:HIV VLPs. Importantly, it did not interfere either with the HIV-1 antibody reactivity targeting sequential and conformational P18I10 and T20 peptides presented on chimeric HPV:HIV VLPs or with the induction of HPV16 L1-specific antibodies in vivo. We observed that chimeric L1:P18I10/L1:T20 VLPs vaccines could induce HPV16- but weak HIV-1-specific antibody responses and elicited HPV16- and HIV-1-specific T-cell responses in BALB/c mice. Moreover, could be a potential booster to increase HIV-specific cellular responses in the heterologous immunization after priming with rBCG.HIVA vaccine. This research work would contribute a step towards the development of the novel chimeric HPV:HIV VLP-based vaccine platform for controlling HPV16 and HIV-1 infection, which is urgently needed in developing and industrialized countries.

Expression of Chimeric HPV-HIV Protein L1P18 in Pichia pastoris; Purification and Characterization of the Virus-like Particles.

Currently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris.

Host-dependent editing of SARS-CoV-2 in COVID-19 patients.

A common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants.

Priming with Recombinant BCG Expressing HTI Enhances the Magnitude and Breadth of the T-Cell Immune Responses Elicited by MVA.HTI in BALB/c Mice.

The use of Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a live vaccine vehicle is a promising approach for HIV-1-specific T-cell induction. In this study, we used recombinant BCG expressing HIVACAT T-cell immunogen (HTI), BCG.HTI2auxo.int. BALB/c mice immunization with BCG.HTI2auxo.int prime and MVA.HTI boost was safe and induced HIV-1-specific T-cell responses. Two weeks after boost, T-cell responses were assessed by IFN-γ ELISpot. The highest total magnitude of IFN-γ spot-forming cells (SFC)/106 splenocytes was observed in BCG.HTI2auxo.int primed mice compared to mice receiving MVA.HTI alone or mice primed with BCGwt, although the differences between the vaccination regimens only reached trends. In order to evaluate the differences in the breadth of the T-cell immune responses, we examined the number of reactive peptide pools per mouse. Interestingly, both BCG.HTI2auxo.int and BCGwt primed mice recognized an average of four peptide pools per mouse. However, the variation was higher in BCG.HTI2auxo.int primed mice with one mouse recognizing 11 peptide pools and three mice recognizing few or no peptide pools. The recognition profile appeared to be more spread out for BCG.HTI2auxo.int primed mice and mice only receiving MVA.HTI. Here, we describe a useful vaccine platform for priming protective responses against HIV-1/TB and other prevalent infectious diseases.

Design Concepts of Virus-Like Particle-Based HIV-1 Vaccines.

Prophylactic vaccines remain the best approach for controlling the human immunodeficiency virus-1 (HIV-1) transmission. Despite the limited efficacy of the RV144 trial in Thailand, there is still no vaccine candidate that has been proven successful. Consequently, great efforts have been made to improve HIV-1 antigens design and discover delivery platforms for optimal immune elicitation. Owing to immunogenic, structural, and functional diversity, virus-like particles (VLPs) could act as efficient vaccine carriers to display HIV-1 immunogens and provide a variety of HIV-1 vaccine development strategies as well as prime-boost regimes. Here, we describe VLP-based HIV-1 vaccine candidates that have been enrolled in HIV-1 clinical trials and summarize current advances and challenges according to preclinical results obtained from five distinct strategies. This mini-review provides multiple perspectives to help in developing new generations of VLP-based HIV-1 vaccine candidates with better capacity to elicit specific anti-HIV immune responses.

Designing Chimeric Virus-like Particle-based Vaccines for Human Papillomavirus and HIV: Lessons Learned.

Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not contain any genetic material so that they are not infectious. VLPs maintain the same antigenic conformation to the original virus, and they could be a better vaccine candidate than live-attenuated and inactivated vaccines. In addition, compared to other subunit vaccines such as soluble protein, VLPs can stimulate both innate and adaptive immune responses effectively and safely against several pathogens by the closer morphology to its native virus. They have already been licensed as vaccines against Hepatitis B virus, human papillomavirus (HPV), and several veterinary diseases. Moreover, it has been investigated to prevent other viral infections including HIV. While HIV VLP-based vaccines have been studied over 35 years, none of them has been successful enough to reach even Phase III clinical trials. In this review, we summarize: (i) general features of VLPs; (ii) epidemiological data and current status of vaccine research and development on HPV and HIV; and (iii) previous studies held on HPV VLPs, HIV VLPs, and chimeric HPV/HIV VLPs including production methods and different animal immunization assays. Furthermore, we review present state of human clinical trials with VLPs and consider the potential to develop a successful preventive HIV vaccine using HPV VLP models. Finally, we discuss the benefits, limitations, and challenges of developing chimeric VLP-based HPV/HIV vaccines with recent findings, critical issues to improve VLP-based vaccines, and hot topics for the next 5 years to join the global effort to fight against these two pathogens.

Recombinant BCG Expressing HTI Prime and Recombinant ChAdOx1 Boost Is Safe and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice.

Despite the availability of anti-retroviral therapy, HIV-1 infection remains a massive burden on healthcare systems. Bacillus Calmette-Guérin (BCG), the only licensed vaccine against tuberculosis, confers protection against meningitis and miliary tuberculosis in infants. Recombinant BCG has been used as a vaccine vehicle to express both HIV-1 and Simian Immunodeficiemcy Virus (SIV) immunogens. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HTI.int, expressing the HIVACAT T-cell immunogen (HTI). The plasmid was transformed into a lysine auxotrophic Mycobacterium bovis BCG strain (BCGΔLys) to generate the vaccine BCG.HTI2auxo.int. The DNA sequence coding for the HTI immunogen and HTI protein expression were confirmed, and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that the vaccine was stable in vitro for 35 bacterial generations, and that when delivered in combination with chimpanzee adenovirus (ChAd)Ox1.HTI in adult BALB/c mice, it was well tolerated and induced HIV-1-specific T-cell responses. Specifically, priming with BCG.HTI2auxo.int doubled the magnitude of the T-cell response in comparison with ChAdOx1.HTI alone while maintaining its breadth. The use of integrative expression vectors and novel HIV-1 immunogens can aid in improving mycobacterial vaccine stability as well as specific immunogenicity. This vaccine candidate may be a useful tool in the development of an effective vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens.

Priming With Recombinant BCG Expressing Novel HIV-1 Conserved Mosaic Immunogens and Boosting With Recombinant ChAdOx1 Is Safe, Stable, and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice.

BCG is currently the only licensed vaccine against tuberculosis (TB) and confers protection against meningitis and miliary tuberculosis in infants, although pulmonary disease protection in adults is inconsistent. Recently, promising HIV-1 immunogens were developed, such as the T-cell immunogens "tHIVconsvX," designed using functionally conserved protein regions across group M strains, with mosaic immunogens to improve HIV-1 variant match and response breadth. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVconsvXint, expressing the immunogens HIVconsv1&2. This expression vector used an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate vaccines BCG.HIVconsv12auxo.int and BCG.HIVconsv22auxo.int. The DNA sequence coding for the HIVconsv1&2 immunogens and protein expression were confirmed and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that BCG.HIVconsv1&22auxo.int in combination with ChAdOx1.tHIVconsv5&6 were well tolerated and induced HIV-1-specific T-cell responses in adult BALB/c mice. In addition, we showed that the BCG.HIVconsv1&22auxo.int vaccine strains were stable in vitro after 35 bacterial generations and in vivo 7 weeks after inoculation. The use of integrative expression vectors and novel HIV-1 immunogens are likely to have improved the mycobacterial vaccine stability and specific immunogenicity and may enable the development of a useful vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens shortly following birth.

MTBVAC-Based TB-HIV Vaccine Is Safe, Elicits HIV-T Cell Responses, and Protects against Mycobacterium tuberculosis in Mice.

The tuberculosis (TB) vaccine MTBVAC is the only live-attenuated Mycobacterium tuberculosis (Mtb)-based vaccine in clinical development, and it confers superior protection in different animal models compared to the current vaccine, BCG (Mycobacterium bovis bacillus Calmette-Guérin). With the aim of using MTBVAC as a vector for a dual TB-HIV vaccine, we constructed the recombinant MTBVAC.HIVA2auxo strain. First, we generated a lysine auxotroph of MTBVAC (MTBVACΔlys) by deleting the lysA gene. Then the auxotrophic MTBVACΔlys was transformed with the E. coli-mycobacterial vector p2auxo.HIVA, harboring the lysA-complementing gene and the HIV-1 clade A immunogen HIVA. This TB-HIV vaccine conferred similar efficacy to the parental strain MTBVAC against Mtb challenge in mice. MTBVAC.HIVA2auxo was safer than BCG and MTBVAC in severe combined immunodeficiency (SCID) mice, and it was shown to be maintained up to 42 bacterial generations in vitro and up to 100 days after inoculation in vivo. The MTBVAC.HIVA2auxo vaccine, boosted with modified vaccinia virus Ankara (MVA).HIVA, induced HIV-1 and Mtb-specific interferon-γ-producing T cell responses and polyfunctional HIV-1-specific CD8+ T cells producing interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), and CD107a in BALB/c mice. Here we describe new tools to develop combined vaccines against TB and HIV with the potential of expansion for other infectious diseases.

Advances and challenges in recombinant Mycobacterium bovis BCG-based HIV vaccine development: lessons learned.

INTRODUCTION: Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome, tuberculosis, and malaria are responsible for most human deaths produced by infectious diseases worldwide. Vaccination against HIV requires generation of memory T cells and neutralizing antibodies, mucosal immunity, and stimulation of an innate immune responses. In this context, the use of Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a live vaccine vehicle is a promising approach for T-cell induction. AREAS COVERED: In this review, we provide a comprehensive summary of the literature regarding immunogenicity studies in animal models performed since 2005. Furthermore, we provide expert commentary and 5-year view on how the development of potential recombinant BCG-based HIV vaccines involves careful selection of the HIV antigen, expression vectors, promoters, BCG strain, preclinical animal models, influence of preexisting immunity, and safety issues, for the rational design of recombinant BCG:HIV vaccines to prevent HIV transmission in the general population. EXPERT COMMENTARY: The three critical issues to be considered when developing a rBCG:HIV vaccine are codon optimization, antigen localization, and plasmid stability in vivo. The use of integrative expression vectors are likely to improve the mycobacterial vaccine stability and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth.