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HIV-1 Vaccines Focused on Production of Broadly Neutralizing Antibodies to Inhibit Transcytosis of Virus in Epithelial Cells
The human immunodeficiency virus type 1 (HIV-1) is a lentivirus (subgroup of retroviruses) that causes HIV infection and can result in acquired immunodeficiency syndrome (AIDS). HIV infection leads to progressive failure of the immune system which allows opportunistic infections and cancers to develop. Without access to treatment, the estimated survival time after infection is estimated to be 10-14 years, depending on the HIV subtype. There are 36.7 million people currently living with HIV/AIDS, of which children make up 1.8 million. According to the CDC last year in 2016, there were 2.1 million new cases, and 1.1 million died from AIDS-related illnesses in the same year(1). These numbers are shocking considering the fight against HIV/AIDS has taken full swing three decades ago, clearly stating to how important new prevention methods are a necessity. The majority of those affected by HIV are in low and middle income countries. Sub-Saharan Africa is the most affected region and accounted for 66% of all new HIV infections in 2016 and an estimated 25.6 million people currently living with HIV(2). Although worldwide diagnostic rates and those receiving treatment has improved, it is still an unmet challenge especially for those in resource-limiting regions of the world. The development of an effective and safe vaccine for neonates and/or adults would be the best solution for preventing infection and reducing the severity of HIV-related diseases.
Various prevention strategies such as scaling up highly active antiretroviral therapy, large scale circumcision programs, pre- and post- exposure prophylaxis for high risk individuals, topical microbicides have contributed substantially to the reduction in global HIV incidence and mortality. However, the benefits of non-vaccine prevention strategies are often overshadowed by factors such as inadequate financial and human resources, limited compliance and poor adherence(1). For these reasons an HIV vaccine remains critical in combating the current HIV pandemics. This paper gives a brief introduction into the pathogens and the recent advances in HIV vaccine development will be reviewed.
HIV is approximately 100 nm icosahedral structure, with 72 external spikes that are formed by two major envelope glycoproteins gp120 (essential for virus entry and attachment) and gp41 (essential for infection and highly conserved). It has a cone-shape core surrounded by lipid matrix containing key surface antigens and glycoproteins. The viral capsid is composed of viral protein p24 and contains 2 copies of non-covalently linked, positive-sense single stranded RNA. HIV has three main structural genes found in all retroviruses, Group Specific Antigen (Gag), Envelope (Env) and Polymerase (Pol). Gag contains 1500 nucleotides and encodes for four separate proteins which form the building blocks for the viral core. Pol also encodes for four separate proteins, of which Reverse Transcriptase is the most important. Reverse transcriptase performs a unique job of copying the virus’ RNA genome into DNA. The Env gene in HIV encodes a single protein, gp160. When gp160 is synthesized it travels to the cell surface and enzymes cleave it to produce gp120 (located on the outside of the membrane) and gp41 (located on the inside of the membrane)(3).
Mechanism of Infection
Once inside the body, HIV attaches itself to a susceptible host which contains the CD4 receptor such as helper T cells, macrophages, monocytes, B cells, microglial brain cells and Langerhan cells. The virus binds to the CD4 receptor via gp120 and needs the co-receptors CCR5 or CXCr4 in order to tightly bind which causes an irreversible conformational change to gp41, so that the membrane can fuse with that of the host cell. The actual fusion of the membrane occurs within minutes by pore formation and releases the viral core into the cytoplasm of the host cell. The viral core disassembles and a double stranded viral DNA is then formed by the virus’ reverse transcriptase enzyme. Related but distinct viral variants/mutations can be generated during this process since reverse transcriptase is error prone and has no proofreading activity(4).
The double stranded viral DNA other proteins makes the pre-integration complex. The viral DNA then enters the nucleus and integrates itself into a gene rich, transcriptionally active domains of the host’s chromosomal DNA. The hosts cell then begins transcription creating single-stranded RNA known as viral messenger RNA and progeny virion RNA. New viral RNA is used as genomic RNA and to make viral proteins after undergoing translation. The new viral RNA and proteins move to the cell surface for assembly and a new, immature HIV forms. The virus buds off as a new HIV virus and matures by protease cleaving and releasing individual HIV proteins, which can now infect other cells. HIV infection induces cell death by, pyroptosis, apoptosis of itself or of uninfected bystander, direct viral killing of the cell or killing by CD8+ T cells(4, 5).
After primary infection, acute viremia occurs and it is widely disseminated, predominantly to lymphoid tissues. During the early viremic phase, the virus is trapped within the processes of follicular dendritic cells in the germinal centers of lymphoid tissue. The lymphoid tissue is the major reservoir for the site of persistent viral replication, even in the early infection phase where the CD4+ T cell count is only moderately decreased. Virus continues to be trapped by follicular dendritic cells in the germinal centers of the lymph nodes, initiating continuous immune stimulation and constant exposure to possible infection of CD4+ T cells that reside in or are migrating through the lymph nodes(3). In this regard, recent studies have shown that the HIV that is trapped on the follicular dendritic cells is infectious for CD4+ T cells, even though the virions are coated with neutralizing antibodies(6). Thus, the mechanisms operable in an appropriate immune response to HIV, particularly activation of the immune system, are ironically the same mechanisms that propagate HIV infection and lead to the ultimate destruction of lymphoid tissue and to profound immunosuppression. In this setting of persistent viral replication, progressive deterioration of immune function usually occurs, ultimately resulting in profound immunosuppression and clinically apparent disease(5).
It has long been recognized that infection with HIV is characterized not only by development of profound immunodeficiency but also by sustained and dramatic immune activation. Recent evidence establishes immune activation as a critical underlying mediator of immune dysfunction and immune deficiency. This state of immune activation is manifested both by enhanced expression of phenotypic activation markers on peripheral blood T cells and B cells and by increased plasma levels of inflammatory cytokines; moreover, lymphocytes obtained from HIV-infected persons are more often found in activated phases of the cell cycle(4).
During HIV infection, T lymphocytes often express surface markers of immune activation such as HLA class II molecules and CD38, a membrane-bound adenosine 5′-diphosphate (ADP) ribosyl cyclase. These markers of activation are elevated in direct proportion to the magnitude of HIV replication and some studies have found that the extent of CD38 expression predicts HIV disease course more accurately than do plasma levels of HIV itself. Although a simple explanation for this state of persistent immune activation would be a reflection of HIV-specific T-cell expansion, the frequency of phenotypically activated CD8+ T cells found in HIV-infected subjects is often greater than 80%, substantially exceeding the proportion of cells that can be shown to recognize HIV peptides(7). Thus, a significant proportion of this activation may represent a response to other antigens, or may be an indirect (or bystander) effect of HIV replication. Plasma levels of TNF-alpha, IL-1, and IL-6 are often elevated in later stages of HIV infection, and both TNF and IL-6 levels also are directly correlated with plasma HIV RNA levels. Interestingly, in lymphoid tissue, the primary site of HIV replication, levels of TNF-alpha are not generally increased, although expression of IL-1, IL-2, IL-6, IL-12, and interferon-gamma may be elevated(5).
After the discovery of the causative agent of AIDS with the research it took to understand the virus replication cycle, drug discovery efforts were focused on target inhibition with specific pharmacological agents. The CCR5 or CXCR4 antagonists are antiretroviral drugs that can prevent the viral attachment to the CD4 T-cells. Once the HIV binds to a CD4+ surface receptor, it activates other proteins on the surface of the human cell known as CCR5 and CXCR4 to allow the HIV envelope and CD4 cell membrane to fuse. A second group of drugs can interfere with the fusion process (Fusion/Entry inhibitors)(7). Once inside the susceptible cell, the viral capsid containing the RNA and important enzymes is released into the host cell (Uncoating). A viral enzyme called reverse transcriptase converts its genetic material, HIV RNA into HIV DNA, allowing HIV to enter the CD4 cell nucleus. Reverse transcription can be blocked by Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). The newly formed viral DNA is then integrated with the DNA of the human host cell using a viral enzyme called integrase (Integration). The Integrase inhibitors can block the integration phase. Once integrated into the CD4 cell, the CD4 machinery produces long chains of HIV proteins (replication)(5). A viral enzyme called protease cuts these long chains of proteins into smaller proteins to form the structure of the new HIV particle, including each of the enzymes and proteins needed to repeat the reproductive process. Once the new viral particles are assembled, they bud off the host cell and can infect other cells. Protease inhibitors can block viral assembly(7). When these drugs are taken in combination they are called Antiretroviral Therapy (ART).
Vaccine Clinical Trials
In 2005 a phase 3 study conducted by VaxGen’s AIDS VAXgp120 was published and was the first phase 3 placebo-controlled efficacy study of a vaccine to prevent HIV-1 infection. Over 5000 MSMs were enrolled in this study. The method chosen for this study was a double-blind randomized trial of a recombinant HIV-1 envelope glycoprotein subunit (rgp120) vaccine and was conducted on men who have sex with men and among women of high risk for heterosexual transmission of HIV-1. The patients received 7 injections of either vaccine or placebo over 30 months. The end point was determined to be HIV-1 seroconversion over 36 months(8).
Regrettably when the participants were evaluated it was determined that the vaccine did not prevent HIV-1 infection. Infection rates were 6.7% in vaccines and 7.0% in placebo recipients; vaccine efficacy was estimated as 6% (95% confidence interval, -17% to 24%). They detected the production of neutralizing and CD4 blocking antibody responses in all vaccines assessed for immunogenicity, yet the vaccine was ineffective in preventing HIV-1 infection or in modifying post infection markers(9). This study aimed to target B cells to induce production of neutralizing antibodies that would prevent initial infection or acquisition of the virus, unfortunately the vaccine was not immunogenic enough.
Only four years later an researchers conducted the RV144 clinical trials which was the first HIV-1 vaccine trial to show any efficacy. RV144 was a randomized, multicenter, double blind, placebo controlled efficacy trial of recombinant canary pox vector vaccine done among 16,402 participants in Thailand. During the first year the vaccine efficacy was 60% but over 42 months its efficacy dropped to 31.2% (95% confidence interval, 1.1 to 52.1; p=0.04)(9, 10). These results were modest but showed a nondurable vaccine effect, although key findings were made (1) Plasma concentration of IgGantibody specific for V1V2loop region of envelope gp120 was inversely correlated with the risk for HIV acquisition and (2) IgA antibody to HIV-1 Env was directly correlated with acquisition of infection(9, 11). These results highlight the importance of antibody mediated protection and vaccines which would elicit the induction of antibodies.
Broadly Neutralizing Antibodies (bNAbs)
The Fc domain serves as an envoy by which the adaptive immune system can circulate and distribute pathogen specific antibodies at the site of infection, in order to opsonize and engage effector mechanisms to enable clearance of infected cells and virions. Based on past studies antibodies isotypes immunoglobulin A (IgA) and immunoglobulin G (IgG) have been the most widely utilized in achieving passive immunity and they are prevalent in serum and mucosal surfaces(12, 13). Natural passive transfer occurs with IgG antibodies able to utilize the FcRn to achieve placental transport and IgA antibodies prevalence in breast milk and mucosal surfaces. Since most infections occur via mucosal surfaces, IgA antibodies are strong candidates for HIV protection, since they initiate responses such as phagocytosis, ADCC and cytokine release. Some evidence even shows greater benefits with IgA than with IgG although past passive immunity experiments have focused primarily on IgG.
Series of discoveries related to bNAbs have been made recently. Investigators found that roughly 20% of infected individuals developed bNAbs, antibodies capable of neutralizing a wide range of viral isolates, after 2-3 years of infection(2, 6). However, unlike classical viral infection, these bNAbs do not control the viral replication due to continuously emerging escape viruses. Most bNAbs fall into following groups on the bases of the location on the viral spike of the conserved epitopes that they recognize: CD4-binding site, V1/V2 variable loops, certain exposed glycans, and membrane proximal external region(13, 14).
Their efficacy in preventing infection is yet to be proven in humans; however, nonhuman primate studies with passive transfer of bNAbs have promising results(6, 14). With passive transfer of bNAbs, the studies demonstrated that prevention of viral acquisition among uninfected animals and suppression of viremia among infected animals. Ex vivo studies have shown marked suppression of expression of virus from HIV viral reservoirs by bNAbs. Human in vivo studies have shown decreased levels of circulating virus after administration of bNAbs(10, 15). However, development of bNAbs typically occurred only after many years of HIV infection due to evolutionary hurdles that bNAbs must overcome. Some are autoreactive and clonally deleted to prevent the development of autoimmunity; some have specialized structures, such as long complementary-determining regions that are rare among germline B cells; and others require a high degree of somatic hypermutation.
Vaginal epithelial cells are CD4 negative and express minute amounts of CXCR4 or CCR5 co-receptors and thus have received minimal attention regarding HIV-1 infection and transmission. Even with the epithelial cell layer intact, the virus is still able to enter into epithelial cells and can be trancytosed to regions where it can come in contact with a susceptible host(12, 16). A process called transcytosis is thought to be one of the major mechanisms involved in HIV-1 translocation across the single layer columnar epithelium found in the vaginal endocervix, colon and rectum, all frequent portals of entry via mucosal surfaces. Transcytosis is a transcellular transport of cargo molecules from the apical to the basal end of epithelia by endocytic vesicles. Invading pathogens are met with locally produced secretory immunoglobulin IgA and IgG which protect mucosal surfaces. In order to achieve sterilizing immunity at mucosal sites, antibodies must either prevent entry or block viral infectivity. Previous studies have given evidence that show bNAbs block HIV-1 transcytosis at mucosal sites but it is still heavily debated(15, 17).
A recent study showed that potent bNAbs were unable to block in vitro HIV-1 transcytosis but efficiently inhibited the infectivity of the transcytosed virus(18). They used a well-established in vitro system using HEC-1A cell monolayers, and tested the capacity of potent bNAbs (recombinants IgG and IgA monoclonal antibodies) to inhibit HIV-1 transcytosis. They also used wild-type and envelope deficient viruses, which all translocated at equal rates, showing that HIV-1 spike interactions with epithelia cell surface receptors may not be required for transcytosis. Also no mechanistic evidence was seen to support previous theories of HIV-antibody complexes form that lead to blocking HIV-1 entry at mucosal sites(15, 17). Instead showed that through interaction of IgG/IgA with gel forming mucin glycoproteins in vivo, HIV-antibody complexes get trapped in mucus layers and eventually washed out by draining and genital secretions. Unfortunately the half-lives of the most potent bNAbs is the greatest challenge when dealing with passive transfer and much higher titers of antibodies are needed in order to confer protection(7, 18). Ultimately further research is needed to fully understand the mechanisms behind preventing HIV-1 mucosal transmission by bNAbs and transcytosis is essential in optimizing antibody-mediated immunotherapies and vaccine development.
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