A01 Virology and immunology

Introduction

Human immunodeficiency virus type one (HIV-1) is the main causative agent of Acquired Immunodeficiency Syndrome (AIDS) that has led to approximately 40 million deaths of infected people by the end of 2017. Despite the introduction of combination antiretroviral treatment (also referred as HAART, or highly active antiretroviral therapy) since 1996, HIV-1 pandemic continues to grow with about 36.9 million people currently living with the virus. To win the war against HIV/AIDS, it is essential to understand the history of HIV/AIDS and the latest development in the fields of HIV virology and immunology.

In 1981, a previously unrecognised disease was found primarily among men who have sex with men (MSM) in western countries. This disease was characterised by weight loss, fever, pneumonia, Kaposi’s sarcoma (KS) and other opportunistic infections, a clinical syndrome known for immunodeficiency. The disease was later designated as AIDS. Two years after the initial clinical reports, a new lymphadenopathy-associated virus (LAV) was isolated from a lymph node of a MSM patient and was determined as the causative agent of AIDS by a research team at the Pasteur Institute in France.[1] Thereafter, a distinct AIDS-associated retrovirus, which has about 15% genome diversity from LAV, was obtained by a group of US scientists. In 1986, the International Committee on Taxonomy of Viruses gave these viral isolates a new name: human immunodeficiency virus (HIV). A unique lymphadenopathy-associated virus (LAV-II), which also caused AIDS, was subsequently isolated from two West Africans. Since LAV-II was antigenically more closely related to simian immunodeficiency virus (SIVmac) than previous HIV-1 strains, the new virus was designated as HIV-2. HIV-1 has now led to AIDS pandemic throughout the world while HIV-2 remains endemic in West African countries.

Interestingly, soon after HIV-1 and HIV-2 were identified among AIDS patients, SIVmac was isolated from captive Asian macaques with lymphomas.[2] Since purified SIVmac was found to cause AIDS-like diseases in experimentally infected macaques, this finding provided further support that HIV-1 is the causative agent of AIDS in humans. The origin of HIV was derived from viruses found in wild naturally infected African non-human primate species, with HIV-1 from Chimpanzees and HIV-2 from sooty mangabeys, respectively. Both HIV-1 and HIV-2 infections in humans, therefore, are possible outcomes of zoonotic infections.

HIV genome organisation

HIV particles or virions are about 110 nm in diameter. They consist of a lipid bilayer membrane, called envelope, that surrounds a unique cone-shaped nucleocapsid, when observed under electron microscope.[Box 1.1] Each virion contains two identical copies of single-stranded RNA genome (ssRNA) of about 9-10 kilo-base pairs with positive polarity in terms of protein translation. Like other retroviruses, the HIV genome is flanked by long terminal repeat (LTR) sequences. It encodes three structural genes (gag, pol and env), two regulatory genes (tat and rev) and four accessory genes, (nef, vpr, vif and vpu for HIV-1 or vpx for HIV-2).[Box 1.2] Besides structural proteins, only Vpr and Vpx, not Vif, Vpu and Nef, appear to be assembled into virions.

5’LTR contains a promoter essential for viral gene transcription, while the 3’LTR supplies a polyadenylation signal.[Box 1.2] 5’U3 region has binding sites of transcription factors AP-1, NFAT-1, NF-κB and SP1. 5’R region contains a trans-activation response element (TAR) of Tat protein and the start site of transcription. The inhibition of histone deacetylases that act on LTR may facilitate the activation of viral latency in resting CD4⁺ T cells, and it represents a new potential target for subsequent viral elimination by “Shock and Kill” treatment strategy, which is currently under intensive research.

The gag gene encodes precursor polypeptides for matrix (MA, p17), capsid (CA, p24), nucleocapsid (NC, p7) and p6 proteins.[Box 1.2] The pol gene encodes a precursor protein Pr160^Gag-Pol for functional viral enzymes protease (PR, p10), reverse transcriptase (RT, p66/p51), ribonuclease hybrid (RNase H) and integrase (IN, p32). RT is assembled into virions for reverse transcription and this viral protein can be used as an indicator for measuring viral replication. The env gene encodes the precursor glycoprotein for virion envelope surface glycoprotein (gp120) and transmembrane glycoprotein (gp41), which remain connected by a non-covalent bond to form gp160 trimer structure on viral surface.[Box 1.1] The gp160 trimer is required for viral fusion and entry into host cells.

Two regulatory proteins, Tat and Rev, control LTR-directed gene expression at transcriptional and post-transcriptional levels, respectively. Vif is an antagonist of an innate cellular restriction factor APOBEC3 (a cytidine deaminase) of HIV-1 replication. Vpr modulates the reverse transcription, nuclear import of the pre-integration complex, and viral replication in non-dividing cells. Vpx is unique to HIV-2 and some SIV strains. It is an antagonist of host restriction factor SAMHD1 in myeloid-lineage cells (e.g. dendritic cells). Vpu is unique to HIV-1 and it antagonises the host restriction protein BST-2/tetherin to promote viral release. Nef activates T cells to facilitate viral infectivity while also down-regulates the expression of the CD4 receptor and major histocompatibility complex class I molecules to escape immune surveillance.

Life cycle of HIV

The life cycle of HIV-1 includes cell entry, reverse transcription, integration, transcription and translation, assembly and release. The fourteen steps involved are depicted in Box 1.3. The entry of HIV-1 into host cells starts with the binding of viral envelope protein gp120 to the host CD4 receptor on cell surface. After this binding, gp120 undergoes a conformational change and exposes the co-receptor binding site. The subsequent binding of gp120 with a co-receptor, either CCR5 or CXCR4, induces a gp41 conformation transition which allows its hydrophobic N-terminus fusion epitope to penetrate the attached host cell, mediates membrane fusion between the virus and the cell, followed by the release of viral RNAs and enzymes into the cell. The blockade of functional viral structural proteins and enzymes serves as the major HAART targets for antiretroviral development. In 2018, the fusion epitope was found to be a useful immunogen for inducing broadly reactive neutralising antibodies (bNabs).

The high rate of HIV-1 replication, which produces up to 10⁹-10¹⁰ new virions per day, together with a high mutation rate of approximately 3 x 10^-5 per nucleotide per cycle of replication, lead to the generation of viral quasispecies consisting of a pool of genetically diverse viruses.[3] The high mutation rate of HIV-1 is largely due to the error-prone nature of RT, which lacks the proofreading ability of cellular DNA polymerases. Nowadays, pandemic HIV-1 strains can be diversified into tens of distinct genetic subtypes (http://www.hiv.lanl.gov). Subtype C is apparently the most prevalent, accounting for about half of new infections worldwide. The inter-subtype sequence diversity can reach as high as 35% in env gene. Even for viruses within the same subtypes, their env sequences can differ by as much as 20%. This high level of genetic diversity has posed great challenges for AIDS vaccine development.

HIV transmission

The modes of HIV-1 transmission have been well documented , and include unprotected sex (intercourse without a condom), transfusions of unscreened blood, the use of contaminated needles (most frequently for injecting drug use), and vertical transmission from an infected mothers to infants during pregnancy, childbirth or breastfeeding. HIV-1 transmission via unprotected sexual contacts, however, is the major mode of viral spread in the general population all over the world.

The mucosal surface is the first line of defense to prevent pathogens from entering the body. During homosexual or heterosexual transmission, HIV-1 mainly targets either rectal or vaginal mucosa for entry and early replication. HIV-1 penetrates the intestinal or genital barriers to infect CD4⁺ T cells, mainly Th17 cells, probably with the help of M cells, dendritic cells (DCs), and epithelial cells. M cells are present in tonsils and intestines but not in the genital mucosa. DCs may also facilitate HIV-1 dissemination via virion capture by binding viral gp120 with the cellular DC-SIGN receptor. DCs may then deliver the captured virions to the underlying CD4+ T cells via virological synapses.[4] Epithelial and mast cells may also play a role in HIV-1 mucosal infection. Galactosylceramide expressed on epithelial cells of the small intestine can serve as an alternative receptor for HIV-1 to cross the epithelia barrier through transcytosis. Moreover, after stimulation by HIV-1, mucosal epithelial cells could produce thymic stromal lymphopoietin (TSLP), which is an activator for DCs to recruit target CD4⁺ T cells into mucosal tissues to promote HIV-1 infection.

The mucosal immune system is composed of mucosal-associated lymphoid tissues (MALTs). It contains as much as 80% of lymphocytes in the body and is highly compartmentalised and independent from the systemic immune apparatus. Using the SIV/macaque model, it was found that high viral load and CD4⁺ T cell depletion are first detected in the intestinal lymphoid tissues instead of peripheral blood regardless of the route of infection.[5] Subsequently, it has been shown that CD4⁺ T cell loss in gut-associated lymphoid tissues (GALTs) lasts through all stages of HIV-1 infection and AIDS development, suggesting that GALTs are the predominant and initial sites of SIV/HIV replication soon after viral transmission.

Interestingly, it has been found in the SIV/macaque model that the founder viral populations established in mucosal lymphoid tissues are small at the early infection stage despite exposure to a huge amount of virus. Researchers have also identified a dramatic evolutionary bottleneck for HIV-1 mucosal transmission by demonstrating that the productive infection initiates from a single transmitted/founder (T/F) virus in nearly 80% of heterosexual transmission cases in humans. This finding indicates an opportunity for the control of infection by targeting these initially small and genetically homogeneous foci of infection in the first week of infection. The investigation of HIV-1 sexual transmission has been essential for the development of effective biomedical interventions (e.g. male circumcision and microbicides) for prevention.

HIV immunology

In order to rapidly establish a persistent infection in mucosal lymphatic tissues, HIV-1 must overcome host innate immune defenses at the entry stage of infection. This process reflects a complex interplay between the virus and the host defense.[Box 1.4] Usually, when HIV-1 wins, the virus starts replicating in the mucosa, submucosa, and draining lymphoreticular tissues, but cannot be readily detected in plasma (eclipse phase). This eclipse phase generally lasts 7 to 21 days. Sometimes, viral RNA becomes detectable by sensitive fourth-generation assays in blood as early as 7-14 days after infection, which is the first chance for the diagnosis of HIV-1 infection to confirm the onset of viraemia. HIV-1 load increases subsequently to reach a peak viraemia with 100 million copies/mL at 8-12 weeks post infection, at a time close to the antibody seroconversion phase. The viraemia then rapidly decline to a lower steady level (viral setpoint) or an undetectable level. This rapid decline is due to the host adaptive humoral and cellular immune responses that control viral replication effectively at the acute stage of infection. Despite being effective, unfortunately, the host immunity is unable to clear HIV-1 from the human body completely due to viral integration/latency and rapid virus escape. The viral latency can be established as early as 3 days after infection. Although no severe clinical symptoms appear in infected individuals during this time period, host CD4⁺ T lymphocytes are progressively depleted especially in the gut. Eventually, persistent infection is established as a consequence of productive and lasting HIV-1 replication and the chronic activation of T lymphocytes. With an average of 6-8 years, the host immune system is gradually weakened. Once the CD4⁺ T cell count drops under 200 cells/μl in the blood, most infected individuals develop opportunistic infections by bacteria, viruses, fungi and parasites or of tumors, leading to a diagnosis of AIDS and related deaths (see Chapter A3). About 0.5% cases become long-term nonprogressors (LTNPs) with low viral loads (~10³ RNA copies/ml) and stable CD4⁺ T-cell counts. Some LTNPs even have undetectable viral loads and are defined as ‘elite controllers‘.[6]

Innate immunity

Besides the physical mucosal barrier, some soluble small proteins also contribute to the innate defense against HIV-1 infection. Chemokines such as RANTES, MIP-1α and MIP-1β are the major HIV-suppressive factors produced by T cells. Lactoferrin in breast milk and genital secretions blocks HIV-1 infection from DCs to T cells in vitro. The secretory leucocyte protease inhibitor (SLPI) in human saliva exhibits anti-HIV activity probably as part of the HIV-1 resistance in the oral cavity. Elafin/trappin-2 elevated in the genital tract of female sex workers is potentially associated with protection against HIV-1 acquisition. Moreover, α-defensins and β-defensins are likely associated with protection against HIV-1 sexual transmission. The elevated levels of both α-defensins and β-defensins have been found among HIV-1 highly exposed seronegative (HESN) individuals who are defined as individuals lacking anti-HIV-1 IgG seropositivity or evidence of infection despite frequent exposure to virus and/or repeated high-risk behaviour. It is possible that the release of these soluble factors is simply due to innate immune responses to genital tract inflammation related to ongoing bacterial infections or sexually transmitted diseases.

Natural killer (NK) and DCs are also critical contributors to the host innate immunity against HIV-1 infection. NK cells may recognise target cells that exhibit stress signs and cells that expressed down-regulated level of MHC class I proteins. IFN-γ production by NK cells was elevated in a cohort of HESN individuals exposed to HIV-1 through sexual intercourse with a known HIV-1-infected partner. Moreover, genetic analysis on protective NK KIR receptor genotypes together with increased NK activation and degranulation as measured by CD69 and CD107a indicate that increased NK is associated with better protection from HIV-1 during high-risk activity. In addition, NK activity is influenced directly by soluble cytokines such as IFN-γ, IL-2, IL-12, IL-15, IL-18 and IL-21, which may augment NK cytolytic ability. In this regard, plasmacytoid DCs (pDCs) is required for innate defense against acute viral infection via Toll-like receptor-mediated secretion of IFN-α (e.g. TLR-7 and -9), which helps NK-mediated lysis of HIV-1-infected cells. It, however, remains unknown if innate activation driven by HIV-1 exposure helps the recruitment of CD4⁺ target cells to become a permissive site for viral replication. Moreover, pDCs may contribute to AIDS progression by promoting immune activation and therefore has been suggested recently as a drug target for patient’s treatment.

Humoral immunity

At the acute phase of infection, HIV-1-specific IgG antibodies are usually elicited, which assists the diagnosis of infection. These IgG antibodies, however, are often binding but non-neutralising. Nabs emerge usually too late over the course of infection to prevent the initial burst of HIV-1 replication and the formation of latent reservoirs. Autologous Nabs occur couple months later whereas broadly neutralising antibodies (bNabs) are rarely found until 2-3 years of infection after the co-evolution of viral quasispecies and B cell maturation. With the advancement of single B cell antibody gene cloning technology, however, more than a dozen of novel bNabs have been identified targeting at various regions of HIV-1 env since 2009. By passive immunisation, a HIV-1-specific bNab protects macaques from repeated intravaginal exposure to low doses of a CCR5-tropic simian-human immunodeficency virus (SHIV).[7] Moreover, passive administration of combined bNabs not only achieves maximal neutralization coverage against an extremely large proportion of diverse HIV-1 isolates at a low concentration but also delays viral rebound for months in the absence of HAART. Engineered bi-specific and tri-specific bNabs also display improved breadth and potency. Although passive immunisation using bNabs has great preventive and therapeutic potential, none of the existing vaccination strategies is able to induce equally potent bNabs. To overcome this major barrier to producing a preventive HIV-1 vaccine, env structure-guided and B-cell lineage-based immunogens have been tested actively in preclinical and clinical studies.

Besides IgG, mucosal IgA is considered to be critical for the prevention of HIV-1 transmission through sexual intercourse. The evidence was derived from the activity of IgA purified from mucosal samples of HESN individuals. Specific neutralising activity of the purified IgA from cervicovaginal fluid was found in some HESN’s samples. Moreover, mucosal HIV-1 gp41-specific IgA derived from HESN individuals might block epithelial viral transcytosis and neutralised CD4⁺ cell infection. However, it remains unclear whether or not these IgA responses are truly protective. In the Thai Phase III HIV vaccine clinical trial (RV144), plasma Env-specific IgA was correlated with decreased vaccine efficacy.

Cellular immunity

Antigen-specific cytotoxic CD8⁺ T cells (CTLs) recognise and destroy infected cells that express HIV-1 peptides in the context of MHC class I molecules (also known as human leukocyte antigens, HLAs, in humans) on the surface of nucleated cells. Long-lasting CLT activity specific to HIV-1 antigens requires the establishment of a memory cell pool that proliferates rapidly in response to antigen re-encounter. The first T cell response to transmitted/founder HIV-1 contributes to the initial decline of plasma virus during acute infection. Virus-specific CTL response becomes maximal 7-14 days following the peak of viremia, concurrent with the decline in viral replication.[Box 1.4] During chronic infection, virus-specific CTLs also contributes to viraemia control because antibody-mediated depletion of CD8⁺ T cells results in rapid viral load increase in SIV/macaque models. Although the CTL response is effective, like other lentiviruses, HIV-1 and SIV are highly mutable. Soon after the CTL response is established, mutations in viral genome start to accumulate corresponding to epitopes of CTL recognition. The rapid turnover of HIV-1 populations occurs in an infected patient partially as a result of the interplay between CTLs and virus escape.[8]

Interestingly, LTNPs often target CTL epitopes found in the conserved regions within the viral genome during early infection, and consequently these individuals display a better long-term control of viral replication. Moreover, host genetics may also affect the long-term control of viral replication. For example, the overrepresentation of HLA-B_27, HLA-B_5701, HLAB_5703 and HLA-B_5801 alleles in individuals demonstrate the greatest control of HIV-1.[9] Currently, high frequency of broadly reactive cell-mediated responses is considered critical for controlling the spread and replication of HIV-1. One study indicated that the stimulation of HIV-1-specific CTLs facilitates the elimination of latent viral reservoir after virus reactivation, which has implications for therapeutic cure of HIV-1 infection.[10]

For prevention, high frequency of mucosal T cell responses is essential for preventing local productive infection and eliminating the initial founder populations of infected cells. HIV-specific CD8⁺ T cell responses are found in the genital mucosa of HIV-1-resistant sex workers in the absence of detectable HIV-1 infection, suggesting the protective role of cell-mediated immunity. Moreover, significantly high ratio of antigen-specific CTL/CD4⁺ helper T lymphocytes is likely needed to control HIV-1 or SIV in the mucosal tissues. Thus far, T-cell based vaccine strategies can prevent neither HIV-1 infection in phase III trials nor SIV/SHIV infection in non-human primate models. Nevertheless, recent studies demonstrated in macaques that CMV- and PD1-based T-cell based vaccines achieve SIV/SHIV viraemia control for a prolonged period of time in the absence of HAART, which is useful for functional cure of HIV-1 infection. For an effective vaccine, therefore, it is necessary to elicit high levels of both broadly reactive Nabs and CTLs.

References