Canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor alpha secretion

Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-α) secretion and was significantly blocked in the presence of anti-TNF-α antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-α and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-α secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors

Preparation of clinical-grade recombinant canarypox-human immunodeficiency virus vaccine-loaded human dendritic cells

Preclinical data are reported that support a human immunodeficiency virus (HIV) vaccine strategy using recombinant canarypox-HIV vectors (ALVAC-HIV) to load human dendritic cells (DCs) with HIV antigens. Clinical-grade DCs were infected with good manufacturing practice-grade ALVAC-HIV vaccine constructs. ALVAC infection, HIV gene expression, and DC viability and function were monitored by use of immunohistochemistry, flow cytometry, blastogenesis assays, antigen-specific interferon (IFN)-γ enzyme-linked immunospot assay, and enzyme-linked immunosorbent assay protein detection. The vaccines infected both immature and mature DCs, and intracellular HIV-1 Gag protein was detected within hours. ALVAC-HIV induced DC maturation that was mediated by tumor necrosis factor-α and induced DC apoptosis that was directly related to the length of vaccine exposure. Of importance, the infected DCs remained functional in T cell stimulation assays and induced HIV antigen-specific CD8+ T cell production of IFN-γ from cells of HIV-1-infected individuals. These data support an ongoing HIV vaccine trial comparing conventional vaccine delivery routes with ex vivo vaccine-loaded autologous DCs for immunogenicity in HIV-1-uninfected volunteers

Defining epitope coverage requirements for T cell-based HIV vaccines: Theoretical considerations and practical applications

<p>Abstract</p> <p>Background</p> <p>HIV vaccine development must address the genetic diversity and plasticity of the virus that permits the presentation of diverse genetic forms to the immune system and subsequent escape from immune pressure. Assessment of potential HIV strain coverage by candidate T cell-based vaccines (whether natural sequence or computationally optimized products) is now a critical component in interpreting candidate vaccine suitability.</p> <p>Methods</p> <p>We have utilized an N-mer identity algorithm to represent T cell epitopes and explore potential coverage of the global HIV pandemic using natural sequences derived from candidate HIV vaccines. Breadth (the number of T cell epitopes generated) and depth (the variant coverage within a T cell epitope) analyses have been incorporated into the model to explore vaccine coverage requirements in terms of the number of discrete T cell epitopes generated.</p> <p>Results</p> <p>We show that when multiple epitope generation by a vaccine product is considered a far more nuanced appraisal of the potential HIV strain coverage of the vaccine product emerges. By considering epitope breadth and depth several important observations were made: (1) epitope breadth requirements to reach particular levels of vaccine coverage, even for natural sequence-based vaccine products is not necessarily an intractable problem for the immune system; (2) increasing the valency (number of T cell epitope variants present) of vaccine products dramatically decreases the epitope requirements to reach particular coverage levels for any epidemic; (3) considering multiple-hit models (more than one exact epitope match with an incoming HIV strain) places a significantly higher requirement upon epitope breadth in order to reach a given level of coverage, to the point where low valency natural sequence based products would not practically be able to generate sufficient epitopes.</p> <p>Conclusions</p> <p>When HIV vaccine sequences are compared against datasets of potential incoming viruses important metrics such as the minimum epitope count required to reach a desired level of coverage can be easily calculated. We propose that such analyses can be applied early in the planning stages and during the execution phase of a vaccine trial to explore theoretical and empirical suitability of a vaccine product to a particular epidemic setting.</p

Specific Antibody Production by Blood B Cells is Retained in Late Stage Drug-naïve HIV-infected Africans

Unseparated peripheral blood mononuclear cells (PBMCs) obtained from drug-naïve African individuals living in a context of multi-infections and presenting with high viral load (VL), were cultured in vitro and tested for their ability to produce antibodies (Abs) reacting with HIV-1 antigens. Within these PBMCs, circulating B cells were differentiated in vitro and produced IgG Abs against not only ENV, but also GAG and POL proteins. Under similar experimental conditions, HAART treated patients produced Abs to ENV proteins only. The in vitro antibody production by drug-naïve individuals' PBMCs depended on exogenous cytokines (IL-2 and IL-10) but neither on the re-stimulation of reactive cells in cultures by purified HIV-1-gp 160 antigen nor on the re-engagement of CD40 surface molecules. Further, it was not abrogated by the addition of various monoclonal Abs (mAbs) to co-stimulatory molecules. This suggests that the in vitro antibody production by drug-naïve individuals' PBMCs resulted from the maturation of already envelope and core antigen-primed, differentiated B cells, presumably pre-plasma cells, which are not known to circulate at homeostasy. As in vitro produced Abs retained the capacity of binding antigen and forming complexes, this study provides pre-clinical support for functional humoral responses despite major HIV- and other tropical pathogen-induced B cell perturbations

Identification of Germinal Center B Cells in Blood from HIV-infected Drug-naive Individuals in Central Africa

To better understand the pathophysiology of B cell populations—the precursors of antibody secreting cells—during chronic human immunodeficiency virus (HIV) infection, we examined the phenotype of circulating B cells in newly diagnosed Africans. We found that all African individuals displayed low levels of naive B cells and of memory-type CD27+ B cells, and high levels of differentiated B cells. On the other hand, HIV-infected African patients had a population of germinal center B cells (i.e. CD20+, sIgM-, sIgD+, CD77+, CD138±), which are generally restricted to lymph nodes and do not circulate unless the lymph node architecture is altered. The first observations could be linked to the tropical environment whereas the presence of germinal center B cells may be attributable to chronic exposure to HIV as it is not observed in HIV-negative African controls and HAART treated HIV-infected Europeans. It may impact the management of HIV infection in countries with limited access to HIV drugs and urges consideration for implementation of therapeutic vaccines

DC-SIGN (CD209) Mediates Dengue Virus Infection of Human Dendritic Cells

Dengue virus is a single-stranded, enveloped RNA virus that productively infects human dendritic cells (DCs) primarily at the immature stage of their differentiation. We now find that all four serotypes of dengue use DC-SIGN (CD209), a C-type lectin, to infect dendritic cells. THP-1 cells become susceptible to dengue infection after transfection of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), or its homologue L-SIGN, whereas the infection of dendritic cells is blocked by anti–DC-SIGN antibodies and not by antibodies to other molecules on these cells. Viruses produced by dendritic cells are infectious for DC-SIGN– and L-SIGN–bearing THP-1 cells and other permissive cell lines. Therefore, DC-SIGN may be considered as a new target for designing therapies that block dengue infection

A Double-Blind Randomized Phase I Clinical Trial Targeting ALVAC-HIV Vaccine to Human Dendritic Cells

BACKGROUND: We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone. METHODOLOGY/PRINCIPAL FINDINGS: Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production. CONCLUSIONS/SIGNIFICANCE: ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00013572

Phase I Safety and Immunogenicity Evaluation of MVA-CMDR, a Multigenic, Recombinant Modified Vaccinia Ankara-HIV-1 Vaccine Candidate

We conducted a Phase I randomized, dose-escalation, route-comparison trial of MVA-CMDR, a candidate HIV-1 vaccine based on a recombinant modified vaccinia Ankara viral vector expressing HIV-1 genes env/gag/pol. The HIV sequences were derived from circulating recombinant form CRF01_AE, which predominates in Thailand. The objective was to evaluate safety and immunogenicity of MVA-CMDR in human volunteers in the US and Thailand.MVA-CMDR or placebo was administered intra-muscularly (IM; 10(7) or 10(8) pfu) or intradermally (ID; 10(6) or 10(7) pfu) at months 0, 1 and 3, to 48 healthy volunteers at low risk for HIV-1 infection. Twelve volunteers in each dosage group were randomized to receive MVA-CMDR or placebo (10∶2). Volunteers were actively monitored for local and systemic reactogenicity and adverse events post vaccination. Cellular immunogenicity was assessed by a validated IFNγ Elispot assay, an intracellular cytokine staining assay, lymphocyte proliferation and a (51)Cr-release assay. Humoral immunogenicity was assessed by ADCC for gp120 and binding antibody ELISAs for gp120 and p24. MVA-CMDR was safe and well tolerated with no vaccine related serious adverse events. Cell-mediated immune responses were: (i) moderate in magnitude (median IFNγ Elispot of 78 SFC/10(6) PBMC at 10(8) pfu IM), but high in response rate (70% (51)Cr-release positive; 90% Elispot positive; 100% ICS positive, at 10(8) pfu IM); (ii) predominantly HIV Env-specific CD4(+) T cells, with a high proliferative capacity and durable for at least 6 months (100% LPA response rate by the IM route); (iv) dose- and route-dependent with 10(8) pfu IM being the most immunogenic treatment. Binding antibodies against gp120 and p24 were detectable in all vaccination groups with ADCC capacity detectable at the highest dose (40% positive at 10(8) pfu IM).MVA-CMDR delivered both intramuscularly and intradermally was safe, well-tolerated and elicited durable cell-mediated and humoral immune responses.ClinicalTrials.gov NCT00376090

Expansion of Inefficient HIV-Specific CD8 T Cells during Acute Infection

ABSTRACT Attrition within the CD4 + T cell compartment, high viremia, and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8 + T cell responses gain control over viral replication it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to nearly immeasurably low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8 + T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38 + CD27 − CD8 + T cells characterized by the low expression of the CD8 receptor (CD8 dim ). Interestingly, while high frequencies of HIV-specific CD8 + T cell responses occur within the CD38 + CD27 − CD8 dim T cell population, the minority populations of CD8 bright T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8 dim T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence of CD8 dim T cells, and the size of this population inversely correlates with the acute loss of CD4 + T cells. These data indicate, for the first time, that early CD4 + T cell loss coincides with the expansion of a functionally impaired HIV-specific CD8 dim T cell population less efficient in controlling HIV viremia. IMPORTANCE A distinct population of activated CD8 + T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set point, offering the first immunologic evidence of CD8 + T cell dysfunction during acute infection

Effect of Bamlanivimab vs Placebo on Incidence of COVID-19 Among Residents and Staff of Skilled Nursing and Assisted Living Facilities: A Randomized Clinical Trial

IMPORTANCE Preventive interventions are needed to protect residents and staff of skilled nursing and assisted living facilities from COVID-19 during outbreaks in their facilities. Bamlanivimab, a neutralizing monoclonal antibody against SARS-CoV-2, may confer rapid protection from SARS-CoV-2 infection and COVID-19. OBJECTIVE To determine the effect of bamlanivimab on the incidence of COVID-19 among residents and staff of skilled nursing and assisted living facilities. DESIGN, SETTING, AND PARTICIPANTS Randomized, double-blind, single-dose, phase 3 trial that enrolled residents and staff of 74 skilled nursing and assisted living facilities in the United States with at least 1 confirmed SARS-CoV-2 index case. A total of 1175 participants enrolled in the study from August 2 to November 20, 2020. Database lock was triggered on January 13, 2021, when all participants reached study day 57. INTERVENTIONS Participants were randomized to receive a single intravenous infusion of bamlanivimab, 4200mg (n = 588), or placebo (n = 587). MAIN OUTCOMES AND MEASURES The primary outcomewas incidence of COVID-19, defined as the detection of SARS-CoV-2 by reverse transcriptase–polymerase chain reaction and mild or worse disease severity within 21 days of detection, within 8 weeks of randomization. Key secondary outcomes included incidence of moderate or worse COVID-19 severity and incidence of SARS-CoV-2 infection. RESULTS The prevention population comprised a total of 966 participants (666 staff and 300 residents) who were negative at baseline for SARS-CoV-2 infection and serology (mean age, 53.0 [range, 18-104] years; 722 [74.7%] women). Bamlanivimab significantly reduced the incidence of COVID-19 in the prevention population compared with placebo (8.5%vs 15.2%; odds ratio, 0.43 [95%CI, 0.28-0.68]; P < .001; absolute risk difference, −6.6 [95%CI, −10.7 to −2.6] percentage points). Five deaths attributed to COVID-19 were reported by day 57; all occurred in the placebo group. Among 1175 participants who received study product (safety population), the rate of participants with adverse events was 20.1% in the bamlanivimab group and 18.9% in the placebo group. The most common adverse events were urinary tract infection (reported by 12 participants [2%] who received bamlanivimab and 14 [2.4%] who received placebo) and hypertension (reported by 7 participants [1.2%] who received bamlanivimab and 10 [1.7%] who received placebo). CONCLUSIONS AND RELEVANCE Among residents and staff in skilled nursing and assisted living facilities, treatment during August-November 2020 with bamlanivimab monotherapy reduced the incidence of COVID-19 infection. Further research is needed to assess preventive efficacy with current patterns of viral strains with combination monoclonal antibody therapy