**3. Correlates of protection**

32 Immunodeficiency

The virus neutralization is characterized by the interaction of specific antibodies with the viral envelope spikes. This interferes with virus attachment or viral entry in target cells and results in the inhibition of infection. Only a minority of anti-HIV Env antibodies, at any time, exerts immune pressure by autologous neutralization. However, the virus easily mutates and readily escapes from these potentially protective immune responses [32]. During the chronic course of infection only 20% of the infected individuals will generate broadly neutralizing antibodies (bNAbs) having the ability to neutralize heterologous viruses [33]. In addition to classical neutralization, antibodies can attach to HIV infected cells and kill them via antibody dependent cellular cytotoxicity (ADCC) mediated through

The cellular immune response is the other arm of the adaptive immune system (**figure 1**) and it is crucial to combat viral infections. CD8+ cytotoxic T lymphocytes (CTLs), which eliminate infected cells, play a key role in this process. The initial step involves processing of intracellular antigens by the proteasome. The resulting peptides are then presented together with MHC I molecules on the membrane of infected somatic cells. The peptide-MHC I complex is recognized by precursor cytotoxic CD8+ T lymphocytes (CTLs). Also in that case a CD4+ T cell help, induced by antigen presenting cells, is crucial. In this case so-called Th1 cells, producing IL-2, IFN-γ, and TNF-α, activate and differentiate the CTLs into memory or effector CTLs. Effector CTLs can directly kill infected cells by the production of perforines and granzymes (**figure 2**) [36]. Alternatively, CTLs can induce apoptosis of the infected cells after interaction of Fas ligand on CTLs with Fas receptor on infected T cells [37]. CD8+ T cells also display a non-cytotoxic antiviral activity involving several cytokines, chemokines and a

**Figure 2.** HIV-induced T cell responses. HIV specific CD8+ effector cells produce chemokines and cytokines in order to eliminate infected cells. CD4+ T helper cells help to stimulate both dendritic cells and CD8+ T-cells to maintain a CD8+ T-cell memory response. HIV interferes with this supportive

their Fc moiety and natural killers cells (NK) [34, 35].

yet unidentified soluble CD8+ cell antiviral factor (CAF) [38].

function of CD4+ T-cells.

**2.3. HIV-specific cellular immune response** 

To date no definite biological markers have been unambiguously shown to correlate with patients' ability to control HIV infection by suppressing virus production and eliminating infected cells. Defining these factors would be crucial for the development of preventive and therapeutic vaccination strategies. For that reason, research groups focusing on preventive vaccines carefully study individuals that can avoid infection (exposed seronegatives) or partly control the virus load without the need of HAART.

### **3.1. Natural resistance and genetic factors of the host**

Individuals that have the homozygote deletion Δ32 in co-receptor CCR5 are largely resistant to HIV-1 infection [47]. It has been recently reported that an HIV patient, who received *CCR5* Δ32/Δ32 stem cells for transplantation, remained without viral rebound for several years [48, 49].

Certain intracellular molecules, expressed by the host, can at least partly protect against cellular infection or virus release. The most important factors identified so far are APOBEC3G, APOBEC3F, TRIM5α and tetherin. APOBEC3G is a cytosine deaminase that incorporates adenosine instead of guanosine during synthesis of the viral DNA, which results in defective proviral DNA [50]. In addition APOBEC3G promotes natural killer cellmediated lysis [51]. Individuals expressing large amounts of APOBEC3G have lower viral loads during acute infection phase [52]. TRIM5a binds to the viral capsid, blocking replication early in the viral life cycle [53]. Tetherin interferes with the virion release by attaching the mature virions to each other and to the host membrane [54].

Certain polymorphisms in the human leukocyte antigen (HLA type) and T or NK cell receptor can affect the cellular HIV-specific immune responses. It has been shown that human leukocyte antigens B\*27, B\*57 and B\*58 are associated with better control of HIV-1 and slower disease progression [55-57]. Interestingly HLA B\*57 also plays a role in the innate protective immune responses, acting as a natural ligand for inhibitory killer immunoglobulin-like receptors (KIRs). KIR3DL1 and KIR3DS1 are also associated with delay in disease progression [58, 59].

"Wrapped Up" Vaccines in the Context of HIV-1 Immunotherapy 35

**Level of protection Suggested correlates of protection**

Cellular immunity Polyfunctional T cells [66, 69]

**Table 1.** Suggested correlates of protection.

long- term memory cells [78].

**4.1. Prophylactic vaccines** 

**4. Vaccination strategies against HIV-1** 

Host restriction factors High levels of antiviral fator APOBEC3G [51, 52]

KIR3DL1, KIR3DS1 [58, 59]

Avidity of HIV specific T cell responses [65, 66, 74]

High production of TRIM5α [53] Up-regulation of tetherin [54] HLA types B\*27, B\*57, B\*58 [55, 56]

Humoral immunity Neutralizing Abs: IgA antibodies at the mucosal surfaces [60-63]

Proliferative CD4+ and CD8+ T cells [68, 70]

Many infection-related hurdles complicate the development of an HIV vaccine. These include the high genetic variability, the potential of cell-to-cell transmission and other evasion strategies such as down-regulating MHC I in infected cells and latency of the virus [77]. In addition, correlates conferring protection against HIV remain to be established. A number of potential markers have been suggested to prevent or control HIV infection. These comprise: production of high titers of neutralizing antibodies with broad specificities, concomitant HIV-specific activation of CD4+ and CD8+ T cells, polyfunctional T cell responses (production of several immune mediators by the same T cell) and induction of

Prophylactic vaccines rely on the production of antibodies that bind to free virus particles thereby preventing viral entry into host cells (defined as neutralization) and thus block infection. Vaccines are designed to mimic natural infections, by using live-attenuated virus (measles, mumps), chemically inactivated virus (polio) or recombinant subunits of the virus (Hepatitis B). There is circumstantial evidence that neutralizing antibodies could play a role in the protection against HIV. HIV-neutralizing IgA antibodies have been isolated in frequently exposed individuals, who remained uninfected [62, 79]. In addition, passive immunization with several HIV neutralizing IgG monoclonal antibodies protects macaques against infectious SHIV (simian immune deficiency virus with an HIV envelope) [80, 81]. Although attenuated SIV vaccines provided some level of protection against super-infection in macaques, attenuated HIV is considered too risky to be ever tried in humans [82]. Therefore, much effort has been invested in the development of subunit vaccines that could elicit production of neutralizing antibodies. It should be taken into account that broadly neutralizing antibodies (bNAbs) that inhibit also heterologous viruses *in vitro*, can be detected in 20% of naturally infected individuals. This implies that the production of these

Viral factors Deletion in Nef [76] Host genetic factors CCR5 D32/D32 [48, 49]

#### **3.2. Suggested immune correlates**

High and broadly neutralizing antibody titers at the port of the virus entry are likely essential to prevent (new) infections of host cells. Indeed high levels of HIV-neutralizing IgA were detected at mucosal surfaces of some exposed seronegative individuals [60-62]. Moreover, in several models of transmission, passive immunization with neutralizing monoclonal antibodies could protect macaques from infection. In contrast, once infection has been established, neutralizing antibodies seem to be unable to control the virus spread [63]. In order to eliminate infected cells, strong CD8+ T cell responses seem to be of importance. As already discussed, most infected individuals show strong CD8+ T cell responses in reaction to the first viraemia peak, resulting in a decline of the viral load in early infection. Unfortunately, in most cases (except for elite controllers) these responses are not able to maintain full control, mainly due to iterative immune escape [39] and chronic immune activation [23], ultimately resulting in T cell exhaustion [64]. In contrast, HIVspecific CD8+ T cells preserve their function and new effective CD8+ T cell responses can arise against viral escape variants in elite controllers [55]. Additionally, a strong avidity of the T cell receptor for the epitope-MHC-I-complex has been shown to promote polyfunctional CD8+ T cells [65] and to initiate more rapid lysis of the target cell [66, 67]. Furthermore, the presence of polyfunctional CD8+ T cells, that have the capacity to exert different effector functions by producing IFN-γ, TNF-α, IL-2, MIP-1β, perforines and/or granzymes and to proliferate upon antigen stimulation, has been associated with the "controller" status [68, 69]. Another important observation came from the study of Geldmacher and colleagues who reported that responses directed against Gag epitopes are dominant and potentially protective in long term non progressors and elite controllers [70]. One of the reasons for this observation could be escape mutations, in particular HLArestricted epitopes of Gag, that come at a cost of great loss in viral fitness [71-73].

As already explained, maturation and differentiation of CD8+ T cells into functional memory and effector subsets are also dependent on functional CD4+ T helper cells. The remaining CD4+ T cells, after massive depletion during acute infection, need to be polyfunctional by producing at least both IFN-γ an IL-2 in order to proliferate upon antigen stimulation [41] and provide help to CTL. This Th1 function is impaired in HIV non-controllers [74].

Unfortunately, none of these factors can truly predict protection against HIV infection [75]. Therefore, at present it seems wise to conclude that all potential correlates (**table 1**) should be taken into account while designing HIV therapies. This includes the preservation of functional Th1 HIV-specific CD4+ T cells and the availability of central memory and memory effector HIV-specific CD8 T cells, with strong avidity for particular difficult-to-mutate epitopes. In addition also a broad functional activity, including production of several effector cytokines and lytic factors are important to result in high and broad HIVsuppressive immune responses [75].


**Table 1.** Suggested correlates of protection.

34 Immunodeficiency

delay in disease progression [58, 59].

**3.2. Suggested immune correlates** 

suppressive immune responses [75].

immunoglobulin-like receptors (KIRs). KIR3DL1 and KIR3DS1 are also associated with

High and broadly neutralizing antibody titers at the port of the virus entry are likely essential to prevent (new) infections of host cells. Indeed high levels of HIV-neutralizing IgA were detected at mucosal surfaces of some exposed seronegative individuals [60-62]. Moreover, in several models of transmission, passive immunization with neutralizing monoclonal antibodies could protect macaques from infection. In contrast, once infection has been established, neutralizing antibodies seem to be unable to control the virus spread [63]. In order to eliminate infected cells, strong CD8+ T cell responses seem to be of importance. As already discussed, most infected individuals show strong CD8+ T cell responses in reaction to the first viraemia peak, resulting in a decline of the viral load in early infection. Unfortunately, in most cases (except for elite controllers) these responses are not able to maintain full control, mainly due to iterative immune escape [39] and chronic immune activation [23], ultimately resulting in T cell exhaustion [64]. In contrast, HIVspecific CD8+ T cells preserve their function and new effective CD8+ T cell responses can arise against viral escape variants in elite controllers [55]. Additionally, a strong avidity of the T cell receptor for the epitope-MHC-I-complex has been shown to promote polyfunctional CD8+ T cells [65] and to initiate more rapid lysis of the target cell [66, 67]. Furthermore, the presence of polyfunctional CD8+ T cells, that have the capacity to exert different effector functions by producing IFN-γ, TNF-α, IL-2, MIP-1β, perforines and/or granzymes and to proliferate upon antigen stimulation, has been associated with the "controller" status [68, 69]. Another important observation came from the study of Geldmacher and colleagues who reported that responses directed against Gag epitopes are dominant and potentially protective in long term non progressors and elite controllers [70]. One of the reasons for this observation could be escape mutations, in particular HLA-

restricted epitopes of Gag, that come at a cost of great loss in viral fitness [71-73].

and provide help to CTL. This Th1 function is impaired in HIV non-controllers [74].

As already explained, maturation and differentiation of CD8+ T cells into functional memory and effector subsets are also dependent on functional CD4+ T helper cells. The remaining CD4+ T cells, after massive depletion during acute infection, need to be polyfunctional by producing at least both IFN-γ an IL-2 in order to proliferate upon antigen stimulation [41]

Unfortunately, none of these factors can truly predict protection against HIV infection [75]. Therefore, at present it seems wise to conclude that all potential correlates (**table 1**) should be taken into account while designing HIV therapies. This includes the preservation of functional Th1 HIV-specific CD4+ T cells and the availability of central memory and memory effector HIV-specific CD8 T cells, with strong avidity for particular difficult-to-mutate epitopes. In addition also a broad functional activity, including production of several effector cytokines and lytic factors are important to result in high and broad HIV-
