The Question of HIV Vaccine: Why Is a Solution Not Yet Available?

Ever since its discovery, human immunodeficiency virus type 1 (HIV-1) infection has remained a significant public health concern. The number of HIV-1 seropositive individuals currently stands at 40.1 million, yet definitive treatment for the virus is still unavailable on the market. Vaccination has proven to be a potent tool in combating infectious diseases, as evidenced by its success against other pathogens. However, despite ongoing efforts and research, the unique viral characteristics have prevented the development of an effective anti-HIV-1 vaccine. In this review, we aim to provide an historical overview of the various approaches attempted to create an effective anti-HIV-1 vaccine. Our objective is to explore the reasons why specific methods have failed to induce a protective immune response and to analyze the different modalities of immunogen presentation. This trial is registered with NCT05414786, NCT05471076, NCT04224701, and NCT01937455.

The initial interplay between HIV and mucosal innate immunity.

Human Immunodeficiency Virus (HIV) is still one of the major global health issues, and despite significant efforts that have been put into studying the pathogenesis of HIV infection, several aspects need to be clarified, including how innate immunity acts in different anatomical compartments. Given the nature of HIV as a sexually transmitted disease, one of the aspects that demands particular attention is the mucosal innate immune response. Given this scenario, we focused our attention on the interplay between HIV and mucosal innate response: the different mucosae act as a physical barrier, whose integrity can be compromised by the infection, and the virus-cell interaction induces the innate immune response. In addition, we explored the role of the mucosal microbiota in facilitating or preventing HIV infection and highlighted how its changes could influence the development of several opportunistic infections. Although recent progress, a proper characterization of mucosal innate immune response and microbiota is still missing, and further studies are needed to understand how they can be helpful for the formulation of an effective vaccine.

Identification of a novel mutation involved in colistin resistance in Klebsiella pneumoniae through Next-Generation Sequencing (NGS) based approaches.

The spread of multidrug-resistant (MDR) K. pneumoniae carbapenemase-producing bacteria (KPC) is one of the most serious threats to global public health. Due to the limited antibiotic options, colis- tin often represents a therapeutic choice. In this study, we performed Whole-Genome Sequencing (WGS) by Illumina and Nanopore platforms on four colistin-resistant K. pneumoniae isolates (CoRKp) to explore the resistance profile and the mutations involved in colistin resistance. Mapping reads with reference sequence of the most com- mon genes involved in colistin resistance did not show the presence of mobile colistin resistance (mcr) genes in all CoRKp. Complete or partial deletions of mgrB gene were observed in three out of four CoRKp, while in one CoRKp the mutation V24G on phoQ was identified. Complementation assay with proper wild type genes restored colistin susceptibility, validating the role of the amino acid substitution V24G and, as already described in the literature, confirming the key role of mgrB alterations in colistin resistance. In conclusion, this study allowed the identification of the novel mutation on phoQ gene involved in colistin resistance phenotype.

Proper Selection of In Vitro Cell Model Affects the Characterization of the Neutralizing Antibody Response against SARS-CoV-2.

(1) Background: Our aim is the evaluation of the neutralizing activity of BNT162b2 mRNA vaccine-induced antibodies in different in vitro cellular models, as this still represents one of the surrogates of protection against SARS-CoV-2 viral variants. (2) Methods: The entry mechanisms of SARS-CoV-2 in three cell lines (Vero E6, Vero E6/TMPRSS2 and Calu-3) were evaluated with both pseudoviruses and whole virus particles. The neutralizing capability of sera collected from vaccinated subjects was characterized through cytopathic effects and Real-Time RT PCR. (3) Results: In contrast to Vero E6 and Vero E6/TMPRSS2, Calu-3 allowed the evaluation of both viral entry mechanisms, resembling what occurs during natural infection. The choice of an appropriate cellular model can decisively influence the determination of the neutralizing activity of antibodies against SARS-CoV-2 variants. Indeed, the lack of correlation between neutralizing data in Calu-3 and Vero E6 demonstrated that testing the antibody inhibitory activity by using a single cell model possibly results in an inaccurate characterization. (4) Conclusions: Cellular systems allowing only one of the two viral entry pathways may not fully reflect the neutralizing activity of vaccine-induced antibodies moving increasingly further away from possible correlates of protection from SARS-CoV-2 infection.

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection.

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent but Neuropilin-1-dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy.

SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.

Fast inactivation of SARS-CoV-2 by UV-C and ozone exposure on different materials.

The extremely rapid spread of the SARS-CoV-2 has already resulted in more than 1 million reported deaths of coronavirus disease 2019 (COVID-19). The ability of infectious particles to persist on environmental surfaces is potentially considered a factor for viral spreading. Therefore, limiting viral diffusion in public environments should be achieved with correct disinfection of objects, tissues, and clothes. This study proves how two widespread disinfection systems, short-wavelength ultraviolet light (UV-C) and ozone (O3), are active in vitro on different commonly used materials. The development of devices equipped with UV-C, or ozone generators, may prevent the virus from spreading in public places.

Weak correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects.

Plenty of serologic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON® SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas® by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we firstly compared two serologic tests on serum samples collected at two different time points from 46 laboratory-confirmed coronavirus disease-2019 (COVID-19) subjects. Secondly, 85 negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8% and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity evaluated for the two tests ranges from 96.5% to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study. These data further shed light on both potentials and possible limitations related to SARS-CoV-2 serology. In this context, great efforts are still necessary for investigating antibody kinetics to develop novel diagnostic algorithms. Moreover, further investigations on the role of neutralizing antibodies and their correlate of protection will be of paramount importance for the development of effective vaccines.

Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects.

Herpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passage in vitro All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading in in vitro post-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.IMPORTANCE Despite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

Entry inhibition of HSV-1 and -2 protects mice from viral lethal challenge.

The present study focused on inhibition of HSV-1 and -2 replication and pathogenesis in vitro and in vivo, through the selective targeting of the envelope glycoprotein D. Firstly, a human monoclonal antibody (Hu-mAb#33) was identified that could neutralise both HSV-1 and -2 at nM concentrations, including clinical isolates from patients affected by different clinical manifestations and featuring different susceptibility to acyclovir in vitro. Secondly, the potency of inhibition of both infection by cell-free viruses and cell-to-cell virus transmission was also assessed. Finally, mice receiving a single systemic injection of Hu-mAb#33 were protected from death and severe clinical manifestations following both ocular and vaginal HSV-1 and -2 lethal challenge. These results pave the way for further studies reassessing the importance of HSV entry as a novel target for therapeutic intervention and inhibition of cell-to-cell virus transmission.

A Biologically-validated HCV E1E2 Heterodimer Structural Model.

The design of vaccine strategies and the development of drugs targeting the early stages of Hepatitis C virus (HCV) infection are hampered by the lack of structural information about its surface glycoproteins E1 and E2, the two constituents of HCV entry machinery. Despite the recent crystal resolution of limited versions of both proteins in truncated form, a complete picture of the E1E2 complex is still missing. Here we combined deep computational analysis of E1E2 secondary, tertiary and quaternary structure with functional and immunological mutational analysis across E1E2 in order to propose an in silico model for the ectodomain of the E1E2 heterodimer. Our model describes E1-E2 ectodomain dimerization interfaces, provides a structural explanation of E1 and E2 immunogenicity and sheds light on the molecular processes and disulfide bridges isomerization underlying the conformational changes required for fusion. Comprehensive alanine mutational analysis across 553 residues of E1E2 also resulted in identifying the epitope maps of diverse mAbs and the disulfide connectivity underlying E1E2 native conformation. The predicted structure unveils E1 and E2 structures in complex, thus representing a step towards the rational design of immunogens and drugs inhibiting HCV entry.

Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein.

OBJECTIVE: The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). DESIGN: Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). RESULTS: In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. CONCLUSIONS: Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool.

Neutralization interfering antibodies: a "novel" example of humoral immune dysfunction facilitating viral escape?

The immune response against some viral pathogens, in particular those causing chronic infections, is often ineffective notwithstanding a robust humoral neutralizing response. Several evasion mechanisms capable of subverting the activity of neutralizing antibodies (nAbs) have been described. Among them, the elicitation of non-neutralizing and interfering Abs has been hypothesized. Recently, this evasion mechanism has acquired an increasing interest given its possible impact on novel nAb-based antiviral therapeutic and prophylactic approaches. In this review, we illustrate the mechanisms of Ab-mediated interference and the viral pathogens described in literature as able to adopt this "novel" evasion strategy.

Anti-hepatitis C virus E2 (HCV/E2) glycoprotein monoclonal antibodies and neutralization interference.

The suggested HCV escape mechanism consisting in the elicitation of antibody (Ab) subpopulations interfering with the neutralizing activity of other Abs has recently been questioned. In particular, it was originally reported that Abs directed against the 436-447 region (epitope II) of HCV/E2 glycoprotein may interfere with the neutralizing Abs directed against the 412-423 region (epitope I) involved in the binding to CD81. In this paper, we investigate on the molecular features of this phenomenon describing an anti-HCV/E2 monoclonal Ab (mAb) (e509) endowed with a weak neutralizing activity, and whose epitope is centered on epitope II. Interestingly, e509 influenced the potent neutralizing activity of AP33, one of the best characterized anti-HCV/E2 mAb, whereas it did not show any interfering activity against two other broadly neutralizing mAbs (e20 and e137), whose epitopes partially overlap with that of e509 and which possibly displace it from the antigen. These data may give a possible clue to interpret the conflicting studies published to date on the mechanism of interference, suggesting the existence of at least two groups of broadly neutralizing anti-HCV/E2 Abs: (i) those whose epitope is focused on the 412-423 CD81-binding region and whose activity may be hampered by other Abs directed against the 436-447 region, and (ii) those directed against CD81-binding regions but whose epitope contains also residues within the 436-447 region recognized by interfering mAbs, thus competing with them for binding. The conflicting results of previous studies may therefore depend on the relative amount of each of these two populations in the polyclonal preparations used. Overall, a better comprehension of this phenomenon may be of importance in the set up of novel mAb-based anti-HCV therapeutic strategies.

HCV proteins and immunoglobulin variable gene (IgV) subfamilies in HCV-induced type II mixed cryoglobulinemia: a concurrent pathogenetic role.

The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved.

Hepatitis C virus (HCV) infection may elicit neutralizing antibodies targeting epitopes conserved in all viral genotypes.

Anti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.