A Review of CCR5 Antibodies Against HIV: Current and Future Aspects

Abstract

HIV is one of the most devastating viral infections the world has ever encountered. Ever since HIV was first identified in the 1980s, it has claimed millions of lives worldwide. There has been tremendous research and development in the diagnosis, prevention and treatment of HIV. Small molecules have been shown to reduce the virus to nondetectable level in human plasma, however, there are reservoirs of latent virus that reemerge if antiretroviral therapy is stopped. There is no vaccine to prevent or cure HIV. A significant amount of research has been reported in the literature regarding antibodies for CCR5, a HIV entry host receptor. This report describes the role of CCR5 antibody in HIV prevention/treatment and how antibody-conjugated nanoparticles could be a future strategy with the potential to effectively eradicate the virus from the human system.

HIV & pathophysiology of HIV binding to host cells

HIV was discovered in early 1981 and since then has infected and claimed millions of deaths worldwide. According to UNAIDS, 36.7 million people are living globally with HIV in 2016 [1]. A total of 1.8 million people became newly infected with HIV in 2017 [1]. The incidence of new HIV infections among adults has declined an estimated 11% in 2016 [1] due to antiretroviral therapy (ART). The introduction of HIV dates back to 1920–1940 period. The phylogenetic and epidemiologic bioinformatics suggested that HIV-1 could have evolved from chimpanzee simian immunodeficiency virus (SIVcpz), which is very closely related to HIV-1 [2]. This cross-species transmission of SIV infection probably occurred in primate handlers most commonly in the context of hunting [3].

HIV is a retrovirus belonging to a ribonucleic acid lentivirus. The envelope (Env) glycoprotein is a trimer contained 81 glycosylated sites on each trimer [4]. Carbohydrates surround the Env making it difficult for host cells to recognize. The entire HIV genome is encoded in the Env glycoprotein. At the end of the trimer are the glycoproteins gp41 and gp120. Gp120 glycoprotein interacts with the CD4⁺ cell receptor and CCR5 coreceptor on the exterior of the cell membrane of T-cells, macrophages, monocytes and dendritic cells [5–7]. By changing the shape of gp120 glycoprotein, it ‘pulls’ the virus to the cell membrane and gp41 fuses the virus to the cell membrane [8]. This allows the virus to deposit its intracellular contents into the cytoplasm in the cell. Subsequently, HIV infection and replication involves several steps [9]. This report emphasizes binding of HIV particle to host cells and how CCR5 monoclonal antibodies (mAb) could be a treatment modality.

Several different body areas contain a high number of CD4⁺ cells expressing CCR5. The GI tract is a preferential site for HIV infection [10]. Macrophages, monocytes and lymphocytes can express the CCR5 receptor as already discussed. Neurons, astrocytes and microglia in the CNS also express this receptor [11]. Other tissues express CCR5 including the epithelium, endothelium, vascular smooth muscle and fibroblasts. Areas of inflammation contain increased numbers of mononuclear cells with CCR5 facilitating transmission of HIV at those sites [12].

Caucasian individuals with a 32-base-pair deletion in the CCR5 receptor gene region (mutant CCR5 -Δ 32 allele) has been shown to provide almost complete protection against HIV-1 infection in homozygous individuals [13–15], which further highlights the importance of CCR5 receptor role in the HIV-1 infection. The central role of CCR5 in HIV-1 infection dictates demand for novel therapies aimed at the CCR5 receptor for controlling the infection and disease progression. The rest of the report will focus on antibodies against CCR5, constraints associated with CCR5 antibody (Ab) application for HIV and future directions of Ab against HIV.

CCR5 antibodies & application: HIV prevention & treatment

CCR5 antibodies block viral entry in uninfected cells and can interrupt transmission of HIV by antibody-dependent cell-mediated cytotoxicity (ADCC) [16]. CCR5 antibodies can stimulate ADCC, which can delay onset of overt disease or control viral replication. ADCC-mediating mAbs may be able to attack latently infected cells after reactivation [17]. Several different mAbs to cellular receptors have been generated targeting both CD4⁺-binding site of gp120 and CCR5 coreceptor [18–25]. Through B-cell isolation and IgG cloning techniques, broadly neutralizing mAb (bnAbs) have been isolated from HIV-1-infected patient [20,22]. The sites for the mAb have been labeled into four categories. These include CD4-binding site on gp120 [26], the glycan-containing region centered on V1/V2 on gp120, the V3 region centered on the N332 glycan of gp120 and the membrane-proximal external region of gp41 [27]. Several of these antibodies are in clinical trials for HIV treatment [28]. By binding the CCR5 receptor and preventing HIV fusion with CCR5⁺ cells, these mAbs could be useful for HIV prevention. However, the main use in the near future for the CCR5 mAb would be for HIV treatment.

CCR5 antagonist maraviroc binds to the hydrophobic pocket formed by seven transmembrane helices of the CCR5 transmembrane receptor [29]. By sitting deep in this pocket within the cell membrane, this small molecule inhibits HIV entry via an allosteric mechanism [30]. Maraviroc only works when patients are infected with R5 virus (virus that uses CCR5 coreceptor). If a patient's virus is dual tropic or uses X4 as the coreceptor, maraviroc is not effective [31]. Synergy has been reported between CCR5 antibodies and maraviroc by both binding to different CCR5 receptor sites [29].

For CCR5 mAb, it is theorized and investigators have studied some antibodies which may drive the viral population toward using CXCR4 coreceptor [32]. This could then make the CCR5 mAb obsolete for HIV treatment. Other investigators are targeting other viral proteins as targets for mAb development [33]. Whether mAb conjugation to nanoparticle (NP) will reduce resistance development would be of interest for resistant viral isolates.

PRO 140 is a humanized IgG4 mAb (PA14) directed to an epitope on the extracellular loop 2 of CCR5 [34]. PRO 140 mAb has proven to offer significant dose-dependent HIV-1 RNA suppression [21,24]. Another mAb, HGS004 (CCR5mAb004) is a human mAb that binds extracellular loop 2 of CCR5 and inhibits HIV-1 entry [23]. This Ab also demonstrated significant antiviral activity when administered to patients infected with CCR-5 tropic HIV-1. PRO 140 and HGS004 antibodies have demonstrated in vitro as well as preliminary in vivo efficacy and have also shown efficacy in initial phases of human clinical trials [35,36]. HGS004 a human mAb against CCR5 dose–response was investigated in HIV-positive patients [23]. Several different doses (0.4–40 mg/kg) intravenously were studied. Patients tolerated single doses well. For the highest dose (40 mg/kg), 40% of patients maintained greater than 1-log reduction in plasma viral RNA for 28 days. HSG004 plasma levels were reported as nonlinear. Of concern in this study was the change in coreceptor tropism to a mixed/dual tropism in 30% of patients receiving the highest dose (40 mg/kg).

Additionally, a cocktail of bnAbs including 3BNC117, b12 and PGT121 has been used in macaque studies to reduce plasma viremia to undetectable levels infected with SHIV-SF162P3 [37]. In these experiments, 10 mg/kg doses were administered on days 0 and 7; each cocktail of bnAbs was compared with saline control-treated macaques. The antibodies reduced plasma viremia an average 3.4 logs and macaques remained undetectable for a median of 56 days. Only when Ab levels were reduced did the virus return to detectable levels. Additionally, when plasma viral load returned to detectable levels, sequence analysis did not demonstrate the development of resistance.

Constraints of mAb for therapeutic applications

Limitations to the use of antibodies are important to consider for the therapeutic application. The major constraints of mAbs are production, pharmacokinetics and safety. In 1975, Kohler and Milstein invented hybridoma technology for the production of mAb [38]. Bacterial, yeast and mammalian cell lines with hybridoma technology are used in the production of different mAbs. Low yield, tedious purification processes and contamination are bottlenecks for production of mAbs. However, several advancements have been made in the cell culture process for better production of mAb [39,40]. Alternative production techniques (i.e., plants) have been engineered to produce mAbs more efficiently than mammalian cell cultures [41].

Ab structure, size and affinity are some of the factors that affect Ab pharmacokinetics [42,43]. An Ab is a complex molecule, in the Fc region, a subdomain responsible for FcRn-binding resulting in an apparent long half-life [44,45]. One of the early reports of CCR5 mAb b12 demonstrated dose-dependent prevention of SHIV in macaques. Here, 25 mg/kg provided 100% efficacy, 5 mg/kg provided 50% efficacy and 1 mg/kg did not provide any protection [46]. This was one of the first reports of dose-dependent efficacy for vaginal prevention of SHIV. mAbs are large molecules that exceed the renal clearance threshold (∼70 kDa), which prevents them from being eliminated through glomeruli. Additionally, due to their protein structure, mAbs cannot be absorbed from GI tract and need to be given as a parenteral formulation. Modified antibodies with small size are rapidly cleared from plasma (half-life: 0.5–30 h) due to a lack of Fc domain [47]. Like small molecules, Ab pharmacokinetics has been affected by concomitant medication. In one study, concomitant drug therapy was shown to increase serum concentrations of delivered antibodies [48].

Unlike small molecules, mAbs are well tolerated in humans. Toxicity related to mAbs can be due to immune reactions of the recipient following recognition of Ab as foreign [49]. Alternatively, mAbs may cause toxicity by interacting with a target antigen in tissues other than the intended use [50]. Overall, the risk of adverse reactions with mAbs is generally low [50].

Future direction: Ab-NP, high-affinity mAb & mAb depo

There is only a single US FDA-approved CCR5 antagonist drug, maraviroc, available for HIV treatment [51,52]. Because of the important role of CD4⁺ cells and CCR5 in the pathogenesis of HIV, an Ab conjugated to an NP delivery system could be highly beneficial considering its safety profile. The nanoformulated mAb could offer extended mean plasma resident time and extend the time between doses. Additionally, an mAb to CCR5 could be a targeting mechanism to activate the latent virus.

The mRNAs encoding VRC01, a bNab against HIV are encapsulated into Lipid NPs and administered to CD34 NSG humanized mice as well as humanized BLT mice [53]. VRC01 Ab dosing as mRNA was attempted using 1.4 mg/kg (30 μg of mRNA-LNP) or 0.7 mg/kg (15 μg of mRNA-LNP) weekly to the mice intravenously (IV). HIV-infected humanized mice receiving either 1.4 or 0.7 mg/kg did not have detectable plasma viral loads 2 weeks after IV administration of HIVSF162 isolate. This represents a new delivery system using nucleoside-modified mRNA as a passive immunotherapy approach.

An interesting study carried out by Tang et al. [54] included loading CD45RO Ab onto a poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA:PEG) copolymer NP with a histone deacetylase inhibitor (HDAC) suberoylanilide hydroxamic acid with or without nelfinavir. These NPs targeted the latent HIV virus in vitro. NPs loaded with suberoylanilide hydroxamic acid+nelfinavir were able to target latently infected CD4⁺ T-cells, activating latent virus and inhibit viral spread. These results demonstrate the potential for both targeting and eliminating latent HIV reservoirs of the NP formulation. Glass et al. produced caveolin-1 NPs functionalized with mAb to CCR5 [55]. These functionalized NPs produced enhanced binding to immune cells, namely CD4⁺ T-cells (4.3- to 43.9-fold). mAb to CCR5 enabled internalization of caveolin-1 NPs by nonphagocytic CD4⁺ T-cells, which has HIV treatment and prevention implications. Finally, Choi et al. were able to conjugate a single-chain variable fragment Ab to target CD7 T-cell receptor with a PLGA NP containing a HDAC used for modulating latent HIV [56]. This Ab fragment conjugation was performed using an oil-in-water sonication method using EDC/NHS reaction producing immuno-NPs. Using flow cytometry, the immune-NPs were delivered to T-cells expressing CD7 receptor with HDAC able to activate latent HIV in vitro. The authors concluded immuno-NPs have the potential to eliminate latent viral reservoirs using a T-cell-specific delivery system. It appears that CCR5 receptor could be used to target latently infected cells particularly in reservoirs (CNS, germinal center).

The efficiency of NP as a carrier system for antiviral agents in HIV to improve the delivery of antiviral agents to intracellular spaces, overcoming pharmacokinetic issues and enhancing activities of drugs has been extensively studied during the last decade [57–62]. Indeed, several NP delivery systems are currently in Phase III trials [60,61]. These NP delivery systems have been designed as parenteral extended release delivery systems with dosing more than 30 days between injections. The NP formulations that are the furthest along in development use a wet-milling system followed by the addition of surfactants to provide extended-release of the wet-milled product. Several trials have demonstrated the potential of the parenteral NP delivery system for HIV therapy [60,61]. Survey of HIV-positive patients reveals a positive response to the overall use of parenteral sustained-release NP delivery systems [63]. The major side effect of this delivery system could be the pain during administration, which most patients tolerate to allow for no ingestion of drug therapy between parenteral injections.

Another delivery system that is being investigated for parenteral NP drug delivery is the use of either oil-in-water or water-in-oil-in-water emulsion methods to formulate the antiretroviral (ARV) drug-loaded NP delivery system [57,64]. Other methods include lipid emulsion methods that have been used for multiple ARV drugs into the nanoformulation [59]. These emulsion methods have been shown to be efficacious in a variety of animal models of HIV infection as well as prevention [59,61,64,65].

Conclusion

HIV prevention is currently successful with ART, but the side effects and daily dosing are the major concerns of ART. In this review, we have discussed the pathophysiology of HIV binding to CD4⁺ cells expressing CCR5 receptors. It is evident from the studies that the efficacy of CCR5 antibodies for HIV treatment and prevention is significant. We have highlighted constraints of mAbs briefly as a measure of caution. Also, highlighted the future directions of Ab–NP conjugation and high-affinity mAbs.

Future perspective

The utility of mAb conjugation to NPs could be another option in the armamentarium for HIV treatment or cure. mAbs to CCR5 receptors could prevent HIV fusion with cells using a mechanism that would be difficult to develop resistance, unlike small molecules for HIV treatment. However, resistance to mAb would need to be investigated since it would be a risk for continued therapy. Additionally, mAb possesses long elimination half-lives and thus may require delivery at infrequent intervals. This is an advantage for HIV patients who incur a lifetime of daily therapy and would prefer infrequent dosing. Continued research into the use of CCR5 mAbs is necessary as there are patients with resistant virus to all major classes of antiretroviral drugs. For these patients, mAb may offer an alternative option for HIV treatment. Further research into appropriate patient selection for this new mAb treatment will be of interest to the HIV community.

Executive summary.

• The use of monoclonal antibody to CCR5 receptors on T-cells and macrophages/monocytes could be a novel treatment modality that would allow for infrequent dosing as well as a barrier to resistance development.

• Conjugation of the monoclonal antibody to a nanoformulation for drug delivery could be a long-acting targeted treatment modality.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.