Introduction

Most people in the world live in poverty-stricken conditions where they are continuously confronted with a plethora of pathogenic organisms—some successfully repelled, some resulting in clinically overt disease, and others resulting in persistent latent infection. The human immune system has evolved to combat these genetically diverse organisms, including viruses, bacteria, and protozoa, through genetically governed responses involving multiple receptors and ligands. Even with clinically overt or persistent infections, most people with an intact immune system ultimately survive most infections.

In marked contrast stands HIV infection. The spread of HIV-1 worldwide represents one of the great challenges to confront host immunity, since the key target is the CD4 T cell lymphocyte, infection and depletion of which severely undermines effective immune responses. As a result, most people who become infected and remain untreated will ultimately succumb to one or more of the large variety of infectious organisms that humans are confronted with on a daily basis. By 2010, there was an estimated 2.4–2.9 million people becoming newly infected with HIV, with 1.8 million dying of AIDS-related causes (http://www.unaids.org, 2011 Global Report).

The ultimate solution to the HIV epidemic relies on the development of an effective vaccine that can be delivered to those at risk. The ability to achieve this elusive goal will be facilitated by a comprehensive understanding of the key immune responses that contribute to protection from infection, or protection from disease progression in those who become infected. In this chapter, we will review the current state of knowledge of what is needed for an AIDS vaccine, first by comparison to effective immune clearance of acute viral infections (such as influenza, rotavirus, or respiratory syncytial virus) as well as acute viral infections followed by latent infection (herpes simplex virus or Epstein–Barr virus). This will allow a foundation for discussing why successful immune mechanisms are not functional in the majority of HIV-1-infected individuals. Much knowledge on the first immune events during acute HIV-1 infection has also accumulated, providing additional clues to the arms of immunity that are triggered upon first encounter with the virus. The field has been shaped by the hypothesis that the initial immune response to HIV infection is translatable to what would be expected or required from a vaccine. We will discuss clues as to what may constitute a protective immune response from disease progression in a small proportion of people who are HIV-1 infected, but can spontaneously contain viral replication to only a few RNA copies. Finally, we will discuss some of the failures and partial successes of recent vaccine trials, which have provided insight into the requirements for potential protective immune mechanisms.

General Principles of an Antiviral Immune Response

The degree to which pathogenic organisms establish productive infections is determined in part by the integrity of epithelial and mucosal cells: skin, respiratory tract, alimentary tract, urogenital tract, and conjunctiva. These regions serve as physical barriers between the exterior and internal environment and any abrasions or lesions will allow potential pathogenic organisms into either the blood or lymphatic circulation. Once these physical barriers have been transcended, there are two major categories of host immune responses, namely innate and acquired immunity.

The innate immune response represents the first line of defense, and serves to rapidly attenuate the impact of most infectious organisms. From an evolutionary perspective, innate immunity shares properties with lower vertebrate mechanisms of engulfment and phagocytosis and the response consists of specialized cells, such as macrophages, natural killer cells, dendritic cells, and polymorphonuclear leukocytes. Infectious organisms that survive the innate immune response, or residues from such a response, are dealt with by the specific acquired immune response.

Acquired immunity has three central tenets: specificity, recognition of protein structures via the interaction of receptors and ligands; diversity, variations in specificity, where multiple receptors interact with different protein structures; and memory, where different T and B cells that have been primed to antigens can be recalled at a subsequent point in time with a more rapid response.

What governs specificity and diversity of the adaptive immune response is the genetic make-up of the host, where genes encoding for the major histocompatibility complex (MHC), T cell receptors (TcR), and immunoglobulins (B cell receptors) dictate how and which regions of the pathogen are encountered by the immune system. The molecules encoded by these genes are central to specificity, diversity, and memory and constitute the internal composition of each individual and is collectively known as “self.” An acquired immune response that results in the successful clearance of an invading organism can be understood by the exquisite difference in recognition between “self” and “non-self.” The ultimate outcome of this process is preservation and survival of the species.

Acquired Immunity to Viral Infections

The immune system consists of parallel blood and lymphatic circulations, ensuring that different cells participating in an immune response can migrate back and forth between non-lymphoid tissue and the different secondary lymphoid structures (such as the spleen and lymph nodes). Bone marrow is the primary lymphoid organ, where the precursors to all mature immunocompetent cells are derived as pluripotential progenitor cells. B and T cells develop into mature immunocompetent cells in the bone marrow and thymus, respectively. T cells that leave the thymus are “naïve” and have yet to encounter invading pathogens.

Lymph nodes are crucial for providing the correct microenvironment and anatomical structures required for initiating an immune response. The micro-anatomical arrangement of the lymph node enables T cells to encounter processed viral proteins presented by specialized antigen presenting cells, which initiates the adaptive immune response. In general terms, if the anatomical arrangement of lymphoid tissue disintegrates due to pathology, the impact will result in disrupted antigen presentation and loss of both B and T cell priming and the inability to provide protective immunity_.

Movement of T cells from one lymphoid region to another allows both CD4 and CD8 T cells to encounter processed antigen in the paracortical region of the lymph node. After engaging and processing antigen in the peripheral tissue, dendritic cells will migrate to lymph nodes, where there is selection of reactive T cells through TcR engagement with viral peptides situated in the binding groove of the human leukocyte antigen molecules on the surface of the antigen presenting cells. This process results in multiple clones of expanded T cells, leading to diversity.

The MHC in humans is known as the human leukocyte antigen (HLA) system and is one of the most polymorphic proteins in the human population. The uniqueness of individuals is partly defined by HLA, where each person has a defined HLA type consisting of pairs of inherited genes. As the sole function of class I and II HLA is to present processed pathogen-derived peptides, or epitopes, to circulating T cells, possible aberrant T cell function and recognition of self, as in autoimmunity, will thus involve the HLA. HLA class I molecules are co-dominantly expressed on antigen presenting cells, and they play an important role in regulating the fitness of the immune system through a process of selecting and presenting immunogenic peptides to CD8 T cells by TcR recognition (Fig. 4.1A). HLA alleles are X-linked and inherited in pairs (heterozygous), and it is noteworthy that in HIV infection, individuals who are homozygous for one or more alleles (inheriting the same HLA allele from both parents) progress more rapidly to AIDS than heterozygotes [1]. Additionally, HLA-B is more polymorphic than HLA-A and HLA-C and the influence of HLA-B is known to have the strongest impact in HIV set point, which is strongly predictive of the rate of progression [2] when compared to HLA-A and HLA-C molecules [3, 4]. The manner by which epitopes are processed and bound by the HLA molecule is highly specific and governed by certain rules associated with the binding motif structures of each HLA and in the correct orientation will be recognized by activated CD8 cytotoxic T lymphocytes (Fig. 4.1A). Sequence changes can occur at anchor positions of targeted epitopes and reduce or interfere with peptide binding to the restricting HLA class I molecule. Moreover, amino acid changes within or immediately adjacent to CD8 T cell epitopes can impede intracellular antigen processing or directly modify the structural interaction between the epitope of HLA class I complex and the TcR of the corresponding CD8 T cells. The ability of viruses to acquire sequence mutations resulting in the loss of recognition by HIV-1-specific CD8 T cells poses a major hurdle for current vaccine efforts.

Figure 4.1A Cells infected by viruses or intracellular bacteria are detected and destroyed by CD8 cytotoxic T lymphocytes. CD8 cytotoxic T lymphocytes are activated in the secondary lymphoid organs and migrate to sites of inflammation where they scan cell surfaces with their T cell receptors (TcR) for recognition of foreign peptide antigens displayed on cell surface HLA class I molecules. HLA class I receptors are constitutively expressed on the surface of all cells except erythrocytes. CD8 cytotoxic T lymphocytes destroy target cells by releasing granzymes and perforin and cell-degrading molecules (with permission from Immunopaedia, http://www.immunopaedia.org).

HLA class II molecules have a more restricted distribution and are expressed only on specific cell types and on T cells after activation. Classically, CD4 T cells provide “help” to the immune response by liberating a series of cytokines important for coordinating cellular activity and inducing activated B cells to become antibody-secreting plasma cells (Fig. 4.1B). First identified in murine models, the multitudinous number of cytokines have been organized into a network model of Th1, Th2, Treg, and Th17 cells. A Th1-type response consists of CD4 T cells liberating a profile of cytokines that direct T cell immunity and involves IL-1, IL-2, IL-6, IL-12, IL-15, TNF-α, and IFN-γ, for example. A Th-2-type response consists of CD4 T cells liberating a profile of cytokines that directs humoral immune responses and is involved in switching on B cell immunity. These cytokines include, among others, IL-4, IL-5, and IL-10. A Th17-type response consists of an IL-17A, IL-17F, and IL-22 profile and is involved in conferring protection against bacteria, fungi, and mycobacteria [5] and to play a role in mucosal defense in the gut. Tregs are CD4⁺CD25^hiFoxP3⁺ and have been shown to downregulate the activation and proliferation of T cells [5]. A balance exists between pro- and anti-inflammatory immune responses imparted by these CD4 subsets for maintaining the integrity and homeostatic balance of cells in the host. Recently, T helper follicular cells that are involved in the development of antibody-producing plasma cells in the germinal centers of lymphoid tissue have been described [6].

Figure 4.1B CD4 helper T lymphocytes stimulate B lymphocytes presenting peptide antigens associated with HLA class II receptors in the T cell zone of secondary lymphoid organs. The CD4 helper T lymphocyte provides activation signals to the B lymphocyte to proliferate and differentiate into antibody-secreting plasma cells. Memory B lymphocytes are also generated for long-term immunity

(with permission from Immunopaedia, http://www.immunopaedia.org).

How do these cells fit together in healthy humans? CD8 T cells make up the smaller proportion of CD3 T cells and are involved in protecting the host from invading pathogens. These cells function by killing virally infected cells, which are marked by the surface expression of HLA class I molecules that present virus-derived epitopes that are typically 8-11 amino acids in length (as described above, Fig. 4.1A). These CD8 T cells function with the help of CD4 T helper cells. The killing potential of CD8 T cells is through either perforin/granzyme or Fas–Fas-L interactions and erupted and effete infected cells are engulfed and processed by dendritic cells; virally derived epitopes are presented via cross-presentation [7] by class II HLA molecules and drive CD4 T cell responses. Typically most (99%) expanded viral antigen-specific T cell effector clones induced in the acute phase of infection will die through apoptosis as the immune response wanes, leaving a small residual population of T effector memory cells that migrate to non-lymphoid tissues or T central memory (TCM) cells that recirculate through the lymphatic system and blood circulation and can be rapidly reactivated upon secondary exposure to viral antigens.

Immune Response to HIV-1 infection

The course of immunological events from the time of transmission can be divided into acute, early, and chronic phases of infection. The greatest challenges HIV presents to the immune system include the selective infection of CD4 T lymphocytes and the extensive viral genetic variability due to mutations.

Is HIV a disease of the mucosal immune system? A number of studies have highlighted the importance of the mucosa in HIV pathogenesis and it is now increasingly being recognized as a disease of the mucosal immune system [8], where vaginal and rectal mucosa are the predominant sites of HIV entry and the gut-associated lymphoid tissue (GALT) is the site of initial HIV replication. During the early phase of simian immunodeficiency virus (SIV) and HIV infection, there is a rapid and widespread massive depletion of activated mucosal CD4 T cells at mucosal sites and this occurs before significant depletion in blood and lymph nodes [9]. Recent evidence confirms that the level of CD4 T cell depletion is far higher than at first anticipated, with 60–80% of memory CD4 T cells depleted during early infection. Thus, within the first few weeks of HIV infection, the virus targets the mucosal immune system and dramatically depletes the CD4 T cells at this site. It has also been shown that HIV targeting of activated CD4 T cells in mucosal tissues persists throughout infection, and not just in acute infection as previously thought. Mucosal tissues are likely to be a major source of viral replication, persistence, and continual CD4 T cell loss in HIV-infected individuals. Reduced CD4 T cell frequencies during chronic HIV infection have also been shown in other mucosal sites such as rectal mucosa [10], male genital tract [11], female genital tract [9], and lung mucosa [12].

Figure 4.2 shows some of the local factors in the mucosal microenvironment that may facilitate HIV replication in mucosal tissues independently from blood: (i) the localized cytokine milieu; (ii) differing inflammatory signals; and (iii) the presence of different immune cell types in these distinct compartments. These factors highlight mucosal sites as critically important in the context of not only understanding HIV pathogenesis but also in terms of being able to possibly dampen or correct the imbalance of pro-inflammatory signals in potential therapeutic or preventive modalities.

Figure 4.2 Epidermal Langerhans cells are a subset of dendritic cells found in the squamous epithelium of the female vagina and male inner foreskin and are the first immune cells to contact HIV during heterosexual contact. They express surface CD207 (langerin) that capture virus by binding to gp120 and induces internalization and degradation of virus. Activated cells migrate to draining lymph nodes for antigen presentation to CD4 T lymphocytes, which can also become infected by surface-bound virus. Langerhans cells also express CD4 and CCR5 and can become infected. Activated Langerhans cells produce pro-inflammatory cytokines IL-1, IL-6, and TNF-α that can cause fever in acute infection (with permission from Immunopaedia, http://www.immunopaedia.org).

During acute HIV infection, there appears to be a hierarchy of systemic immune responses that occur. Figure 4.3 shows a composite schema of the known sequence of immunological responses that occur during infection. After viral transmission (1), where there appears to be a selection of single strain variants at the mucosa [13], there is dissemination (2) of the virus to the lymphoid tissue [14] during the acute phase of infection. Within days after viral transmission, viremia peaks and the downward slope is thought to be a result of a robust cellular immune response leading to initial control (3) of virus [15]. Natural history studies have shown that viral set point is achieved within 6 months of infection and is prognostic of disease outcome, where high levels of viremia are associated with a more rapid course of infection leading to AIDS. It is noteworthy that seroconversion (4), by the detection of anti-Gag binding antibodies, occurs after peak viremia and that detection of neutralizing antibodies occurs only after approximately 3 months post transmission.

Figure 4.3 A typical immune response to untreated HIV infection shows a rapid increase in viremia in the acute phase, which declines to a set point. A decline in CD4 T cells coincides with the increase in viral load. HIV-specific CD8 cytotoxic T lymphocyte responses reduce the viral load and an increase in CD4 T cells is observed. HIV-specific binding antibodies appear after the reduction of viremia, but antibodies are detectable by ELISA only later in acute infection. During chronic infection, CD4 T cells decline slowly and viral load remains stable. Neutralizing antibodies begin to appear around 3 months post infection. Continued HIV replication and immune evasion exhausts the immune system, leading to opportunistic infection and AIDS in most infected people, albeit with varying lengths of time

(with permission from Immunopaedia, http://www.immunopaedia.org).

In addition to virus-specific CD8 T cells, CD4 T cells appear to be critical for immune control. Animal models of chronic viral infections established that virus-specific CD4 T cells play an essential role in maintenance of effective immunity (reviewed in Day and Walker [16]), and the immune response to HIV appears to follow these same requirements. The detection of enhanced proliferation of anti-HIV-specific CD4 T cells in individuals who maintain long-term control of HIV replication [17] and in patients treated for acute infection with potent antiretroviral therapy [17–19] suggest that the function of these cells is central to influencing viral set point and for controlling virus. As discussed, a large number of CD4 T cells are infected in the gut and that the bulk of the CD4 T cell pool resides within lymphoid tissue around the gastrointestinal tract. Direct killing of CCR5 CD4 T cells within the gut [20, 21] and memory CD4 cells in multiple tissues [22] by HIV has been postulated as the main mechanism for CD4 T cell depletion during SIV infection in monkeys, and extrapolations to HIV-induced depletion of CD4 T cells within the human gut have also been made [23]. Figure 4.4 shows the potential scenario of microbial translocation from the gut lumen. Studies of HIV/AIDS pathogenesis have long-focused on the role of CD4 T-cell depletion as a key marker of disease progression [24]. The pathogenesis of HIV infection is now characterized by CD4 T cell immunodeficiency in the context of generalized immune activation and dysregulation, with massive memory CD4 T cell infection and depletion during acute infection. This is followed by gradual loss of remaining CD4 T cells caused by persistent immune hyperactivation. Activation of CD4 T cells results in increased target cells for the virus, excessive apoptosis of uninfected T cells, generalized immune dysfunction, and impaired ability to control HIV replication.