Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

Safety and efficiency modifications of SIV-based integrase-defective lentiviral vectors for immunization.

Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3' polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3' PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3' PPT considerably reduced both expression and immunogenicity of IDLV.

Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy.

Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.

Microbial translocation and T cell activation are modified by direct-acting antiviral therapy in HCV-infected patients.

BACKGROUND: Microbial translocation from the gut lumen has been involved in the pathogenesis of liver damage in hepatitis C virus (HCV) infection. AIM: To investigate the impact of direct-acting antiviral treatment on microbial translocation and T-cell activation, in patients with hepatitis C-related liver disease. METHODS: We enrolled two groups of HCV-infected patients undergoing direct-acting antiviral treatment: patients with fibrosis ≥F3 according to Metavir (Group ≥F3); patients with hepatitis C recurrence after liver transplantation and Metavir ≥F2 (Group Liver Transplantation + ≥F2). All patients were treated with direct-acting antivirals based on ongoing guidelines. Surrogate biomarkers of microbial translocation (plasma concentrations of soluble-CD14, lipopolysaccharide-binding protein and intestinal fatty acid-binding protein) were evaluated at baseline, at first month, at the end of treatment and 3 months later. T-cell activation was measured by expression of CD38+ HLA-DR at the same time points, only in Group ≥F3. RESULTS: There were 32 patients in Group ≥F3 and 13 in Group LT + ≥F2. At baseline, levels of soluble-CD14 and lipopolysaccharide-binding protein were significantly higher in both groups vs healthy controls. Baseline soluble-CD14 correlated with glutamic-oxalacetic transaminase (r = 0.384, P = 0.009) and glutamic-pyruvic transaminase (r = 0.293, P = 0.05). A significant decrease in plasma levels of surrogate microbial translocation biomarkers was observed during and after treatment in the two groups although values were not normalised. In Group ≥F3, CD38+ HLADR+ T-cell expression was significantly decreased by direct-acting antiviral treatment. Relapsers (9%) showed higher soluble-CD14 levels at baseline. CONCLUSION: Surrogate microbial translocation markers and T cell activation are increased in HCV-infected patients with liver fibrosis and decrease during direct-acting antiviral treatment.

Integrase Defective Lentiviral Vector as a Vaccine Platform for Delivering Influenza Antigens.

Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone, or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp + MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by enzyme-linked lectin assay. IFNγ-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed toward HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector.

Reduced Plasma Levels of sCD14 and I-FABP in HIV-infected Patients with Mesalazine-treated Ulcerative Colitis.

BACKGROUND: Microbial translocation (MT) is a shared feature of HIV infection and inflammatory bowel disease (IBD). AIMS: This study was conducted to assess the impact of IBD (and particularly ulcerative colitis, UC) on plasma markers of MT and immune activation in HIV+ subjects. METHODS: A cross-sectional study was conducted in 3 groups of patients: HIV+/UC+(group HIV/UC); HIV+/UC- (group HIV); HIV-/UC+(group UC). Plasma levels of soluble CD14 (sCD14), intestinal fatty acid-binding protein (I-FABP), and endotoxin core antibodies (endoCAB) were measured as plasma markers of MT. Inflammation and immune activation were evaluated by measuring plasma levels of IL-6, IL-21, TNF-alpha, and high-sensitivity C-reactive protein (hs-CRP). T- and B-cells subpopulations were characterized by FACS analysis. RESULTS: Seven patients were enrolled in group HIV/UC, 9 in HIV, and 10 in UC. All HIV-positive patients had plasma values of HIV-1 RNA<37 copies/mL for at least 12 months and good immunological recovery. All patients with UC were treated with oral mesalazine. Markers of MT, immune activation, and inflammation were not increased in subjects with HIV/UC. In fact, they had lower levels of I-FABP (p=0.001) and sCD14 (p=0.007) when compared to other patients groups. Positive correlations were found between I-FABP and sCD14 (r=.355, p=0.076). Frequency of T- and B-cell subsets did not differ among groups. CONCLUSIONS: Our results suggest that UC does not worsen MT, inflammation, or immune activation in HIV-infected subjects. The anti-inflammatory activity of chronic mesalazine administration on intestinal mucosa may contribute to this finding.

Endogenous CCL2 neutralization restricts HIV-1 replication in primary human macrophages by inhibiting viral DNA accumulation.

BACKGROUND: Macrophages are key targets of HIV-1 infection. We have previously described that the expression of CC chemokine ligand 2 (CCL2) increases during monocyte differentiation to macrophages and it is further up-modulated by HIV-1 exposure. Moreover, CCL2 acts as an autocrine factor that promotes viral replication in infected macrophages. In this study, we dissected the molecular mechanisms by which CCL2 neutralization inhibits HIV-1 replication in monocyte-derived macrophages (MDM), and the potential involvement of the innate restriction factors protein sterile alpha motif (SAM) histidine/aspartic acid (HD) domain containing 1 (SAMHD1) and apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family members. RESULTS: CCL2 neutralization potently reduced the number of p24 Gag+ cells during the course of either productive or single cycle infection with HIV-1. In contrast, CCL2 blocking did not modify entry of HIV-1 based Virus Like Particles, thus demonstrating that the restriction involves post-entry steps of the viral life cycle. Notably, the accumulation of viral DNA, both total, integrated and 2-LTR circles, was strongly impaired by neutralization of CCL2. Looking for correlates of HIV-1 DNA accumulation inhibition, we found that the antiviral effect of CCL2 neutralization was independent of the modulation of SAMHD1 expression or function. Conversely, a strong and selective induction of APOBEC3A expression, to levels comparable to those of freshly isolated monocytes, was associated with the inhibition of HIV-1 replication mediated by CCL2 blocking. Interestingly, the CCL2 neutralization mediated increase of APOBEC3A expression was type I IFN independent. Moreover, the transcriptome analysis of the effect of CCL2 blocking on global gene expression revealed that the neutralization of this chemokine resulted in the upmodulation of additional genes involved in the defence response to viruses. CONCLUSIONS: Neutralization of endogenous CCL2 determines a profound restriction of HIV-1 replication in primary MDM affecting post-entry steps of the viral life cycle with a mechanism independent of SAMHD1. In addition, CCL2 blocking is associated with induction of APOBEC3A expression, thus unravelling a novel mechanism which might contribute to regulate the expression of innate intracellular viral antagonists in vivo. Thus, our study may potentially lead to the development of new therapeutic strategies for enhancing innate cellular defences against HIV-1 and protecting macrophages from infection.

Murine granulocyte-macrophage colony-stimulating factor expressed from a bicistronic simian immunodeficiency virus-based integrase-defective lentiviral vector does not enhance T-cell responses in mice.

As a prelude to immunization studies in nonhuman primates, we compared in mice the immunogenicity of a simian immunodeficiency virus (SIV)-based integrase (IN)-defective lentiviral vector (IDLV) encoding the model antigen-enhanced green fluorescence protein (eGFP) in the presence or absence of the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) expressed from an internal ribosomal entry site (IRES) sequence. BALB/c mice were immunized once intramuscularly with IDLV expressing eGFP alone or eGFP and mGM-CSF and immune responses were evaluated up to 90 days from the single intramuscular immunization. Results indicated that the mGM-CSF was unable to improve the magnitude and quality of the immune response against the eGFP transgene in the context of the SIV-based IDLV, as evaluated by enzyme-linked immunosorbent spot (ELISPOT) assays for interferon-γ (IFN-γ) and by intracellular cytokine staining for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α). These findings suggest that for vaccination purposes, the presence of mGM-CSF expressed after the IRES in a SIV-based IDLV system does not favor the improvement of the immunological response against the transgene of interest. Further studies should investigate whether the selection of a different cytokine gene might improve the immune response against the transgene.

Evaluation of HIV-1 integrase inhibitors on human primary macrophages using a luciferase-based single-cycle phenotypic assay.

Macrophages represent an important site for productive infection of HIV-1 and the evaluation of integrase (IN) inhibitors on this cell subset is of fundamental importance. In this report, preclinical evaluation of IN inhibitors on primary human macrophages was attempted successfully using a 96-well microtiter phenotypic assay developed recently for the evaluation of IN inhibitors in a cell-based system by taking advantage of HIV-derived lentiviral vectors expressing luciferase. IN inhibitors were also tested using a lentiviral vector containing an IN with introduced T66I/S153Y mutations, known to affect the activity of azido-group-containing diketo acid (DKA) IN inhibitors. Utilizing different classes of HIV integrase inhibitors against the wild-type IN and the mutant mentioned above, some of the IN inhibitors used were also active on this particular mutant, suggesting that should HIV-1 develop additional or different mutations to become resistant to such anti-IN drugs, new drugs can be developed with a better resistance profile. This assay provides a standardized method for the preclinical evaluation of the efficacy of IN inhibitors on wild-type and mutated IN that can be adapted easily for the evaluation of anti-IN activity on IN sequences derived from patients.

Nonintegrating Lentiviral Vector-Based Vaccine Efficiently Induces Functional and Persistent CD8+ T Cell Responses in Mice.

CD8+ T cells are an essential component of an effective host immune response to tumors and viral infections. Genetic immunization is particularly suitable for inducing CTL responses, because the encoded proteins enter the MHC class I processing pathway through either transgene expression or cross-presentation. In order to compare the efficiency and persistence of immune response induced by genetic vaccines, BALB/c mice were immunized either twice intramuscularly with DNA plasmid expressing a codon-optimized HIV-1 gp120 Envelope sequence together with murine GM-CSF sequence or with a single immunization using an integrase defective lentiviral vector (IDLV) expressing the same proteins. Results strongly indicated that the schedule based on IDLV vaccine was more efficient in inducing specific immune response, as evaluated three months after the last immunization by IFNgamma ELISPOT in both splenocytes and bone marrow- (BM-) derived cells, chromium release assay in splenocytes, and antibody detection in sera. In addition, IDLV immunization induced high frequency of polyfunctional CD8+ T cells able to simultaneously produce IFNgamma, TNFalpha, and IL2.

Transduction of human antigen-presenting cells with integrase-defective lentiviral vector enables functional expansion of primed antigen-specific CD8(+) T cells.

Nonintegrating lentiviral vectors are being developed as a efficient and safe delivery system for both gene therapy and vaccine purposes. Several reports have demonstrated that a single immunization with integration-defective lentiviral vectors (IDLVs) delivering viral or tumor model antigens in mice was able to elicit broad and long-lasting specific immune responses in the absence of vector integration. At present, no evidence has been reported showing that IDLVs are able to expand preexisting immune responses in the human context. In the present study, we demonstrate that infection of human antigen-presenting cells (APCs), such as monocyte-derived dendritic cells (DCs) and macrophages with IDLVs expressing influenza matrix M1 protein resulted in effective induction of in vitro expansion of M1-primed CD8(+) T cells, as evaluated by both pentamer staining and cytokine production. This is the first demonstration that IDLVs represent an efficient delivery system for gene transfer and expression in human APCs, useful for immunotherapeutic applications.

Development and use of SIV-based Integrase defective lentiviral vector for immunization.

Integrase (IN) defective lentiviral vectors have a high safety profile and might prove useful as immunizing agents especially against HIV-1. However, IN defective SIV-based vectors must be developed in order to test their potential in the non-human primate models (NHP) of AIDS. To this aim we tested a novel SIV-based IN defective lentiviral vector for its ability to induce sustained immune responses in mice. BALB/c mice were immunized once intramuscularly with a SIV-based IN defective lentiviral vector expressing the model antigen enhanced green fluorescence protein (eGFP). Immune responses were evaluated 90 days after the injection and compared with those elicited with the IN competent counterpart. The IN defective vector was able to efficiently elicit specific and long-lasting polyfunctional immune responses as evaluated by enzyme-linked immunospot (ELISPOT) assays for interferon-gamma (IFN-gamma) in spleens, bone marrow (BM) and draining lymph nodes, and by intracellular staining (ICS) for IFN-gamma, Interleukin-2 (IL-2) and tumor necrosis factor (TNF-alpha) in both splenocytes and BM cells without integration of the vector into the host genome. This is the first demonstration that an IN defective SIV-based lentiviral vector provides effective immunization, thus paving the way for the construction of IN defective vectors expressing SIV antigen(s) and test their efficacy against a SIV virus challenge in the NHP model of AIDS.

Containment of infection in tat vaccinated monkeys after rechallenge with a higher dose of SHIV89.6P(cy243).

We previously reported that cynomolgus monkeys vaccinated with the human immunodeficiency virus (HIV)-1 Tat protein controlled infection after challenge with the simian human immunodeficiency virus (SHIV) 89.6P(cy243) for up to 2 y of follow-up. To evaluate the breadth of the protective immunity elicited by the Tat protein, the vaccines along with the naïve monkeys were intravenously rechallenged with a fivefold higher dose (50 MID(50)) of the same SHIV-89.6P(cy243). The vaccinated monkeys exhibited a statistically significant and long-lasting reduction of viral replication compared to control monkeys. This effect was associated with a strong anamnestic response to Tat, while responses to Gag and Env were nearly undetectable. Taken together, these data provide further evidence for the usefulness of Tat-based vaccines.

Successful immunization with a single injection of non-integrating lentiviral vector.

We evaluated the ability of an integrase (IN)-defective self-inactivating lentiviral vector (sinLV) for the delivery of human immunodeficiency virus-1 (HIV-1) envelope sequences in mice to elicit specific immune responses. BALB/c mice were immunized with a single intramuscular injection of the IN-defective sinLV expressing the codon optimized HIV-1(JR-FL) gp120 sequence, and results were compared with those for the IN-competent counterpart. The IN-defective sinLV elicited specific and long-lasting immune responses, as evaluated up to 90 days from the immunization by enzyme-linked immunosorbent spot (ELISPOT) and intracellular staining (ICS) for interferon-gamma (IFN-gamma) assays in both splenocytes and bone marrow (BM) cells, chromium release assay in splenocytes, and antibody detection in sera, without integration of the vector into the host genome. These data provide evidence that a single administration of an IN-defective sinLV elicits a significant immune response in the absence of vector integration and may be a safe and useful strategy for vaccine development.

Characterization of alpha-defensins plasma levels in Macaca fascicularis and correlations with virological parameters during SHIV89.6Pcy11 experimental infection.

Alpha-defensins have been shown to inhibit HIV-1 replication in vitro and may contribute to the overall control of viral replication in vivo. In the present work, we quantitatively measured the levels of alpha-defensins in the plasma of healthy and experimentally SHIV-infected Macaca fascicularis (cynomolgus monkeys), an animal model of AIDS pathogenesis and vaccine development. Characterization of physiological plasma alpha-defensins levels was performed in 12 healthy monkeys following longitudinal analysis using an alpha-defensins ELISA kit currently validated for macaque use. Subsequently, alpha-defensins levels were quantitatively measured in 23 cynomolgus monkeys during titration protocols following both the mucosal and systemic routes of infection with the pathogenic SHIV89.6P(cy11). A significant increase in plasma alpha-defensins levels was consistently observed at early time points in all infected animals, regardless of the infection route. Moreover, a positive correlation was observed between viral replication and levels of alpha-defensins during the acute phase of infection. Interestingly, in the animals infected through the mucosal route, alpha-defensins levels remained significantly higher at later time points, up to 19 weeks from the infection, while in cynomolgus infected intravenously, alpha-defensins levels returned to baseline levels by 4 weeks from infection, suggesting that the different route of infection may differently activate the innate immune response.

Evaluation of a self-inactivating lentiviral vector expressing simian immunodeficiency virus gag for induction of specific immune responses in vitro and in vivo.

Humoral and cellular immune responses have been shown to play a fundamental role in controlling simian and/or simian-human immunodeficiency virus (SIV-SHIV) replication in infected macaques. Therefore, the appropriate induction of both compartments of the immune system should be elicited after immunization. In this context, viral vectors have been proven effective in inducing both humoral and cellular immune responses during immunization protocols after direct injection in vivo. Among them, recombinant self-inactivating lentiviral vectors represent a useful strategy for vaccine development because they efficiently transduce and express foreign genes into a wide variety of mammalian cells. Here we report on the development and evaluation of a self-inactivating HIV-based lentiviral vector expressing a codon-optimized SIV Gag sequence (TY2-SIVGagDX), which when used to transduce dendritic cells mediated in vitro expansion of Gag-specific T cells derived from an SHIV-infected cynomolgus monkey, as measured by interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) and (51)Cr release standard assays. To evaluate the ability to elicit specific immune responses in vivo, TY2-SIVGagDX was also employed in a vaccination protocol after a single intramuscular injection in BALB/c mice. Results indicated that the vector was able to efficiently induce both cellular and humoral responses, as measured by IFN-gamma ELISPOT assay and antibody production. These data further confirm that lentiviral vectors encoding viral genes represent an advantageous delivery system for vaccine development.

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.

Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.