Chemokine receptors comprise a large family of seven transmembrane domain G protein-coupled receptors differentially expressed in diverse cell types. Biological activities have been most clearly defined in leukocytes, where chemokines coordinate development, differentiation, anatomic distribution, trafficking, and effector functions and thereby regulate innate and adaptive immune responses. Pharmacological analysis of chemokine receptors is at an early stage of development. Disease indications have been established in human immunodeficiency virus/acquired immune deficiency syndrome and in Plasmodium vivax malaria, due to exploitation of CCR5 and Duffy, respectively, by the pathogen for cell entry. Additional indications are emerging among inflammatory and immunologically mediated diseases, but selection of targets in this area still remains somewhat speculative. Small molecule antagonists with nanomolar affinity have been reported for 7 of the 18 known chemokine receptors but have not yet been studied in clinical trials. Virally encoded chemokine receptors, as well as chemokine agonists and antagonists, and chemokine scavengers have been identified in medically important poxviruses and herpesviruses, again underscoring the importance of the chemokine system in microbial pathogenesis and possibly identifying specific strategies for modulating chemokine action therapeutically. The purpose of this review is to update current concepts of the biology and pharmacology of the chemokine system, to summarize key information about each chemokine receptor, and to describe a widely accepted receptor nomenclature system, ratified by the International Union of Pharmacology, that is facilitating clear communication in this area.

https://pharmrev.aspetjournals.org/content/pharmrev/52/1/145.full.pdf

International Union of Pharmacology. XXII. Nomenclature for Chemokine Receptors

Viral envelope glycoproteins promote viral infection by mediating the fusion of the viral membrane with the host-cell membrane. Structural and biochemical studies of two viral glycoproteins, influenza hemagglutinin and HIV-1 envelope protein, have led to a common model for viral entry. The fusion mechanism involves a transient conformational species that can be targeted by therapeutic strategies. This mechanism of infectivity is likely utilized by a wide variety of enveloped viruses for which similar therapeutic interventions should be possible.

Mechanisms of viral membrane fusion and its inhibition

Induction of broadly cross-reactive neutralizing antibodies is a high priority for AIDS vaccine development but one that has proven difficult to be achieved. While most immunogens generate antibodies that neutralize a subset of T-cell-line-adapted strains of human immunodeficiency virus type 1 (HIV-1), none so far have generated a potent, broadly cross-reactive response against primary isolates of the virus. Even small increments in immunogen improvement leading to increases in neutralizing antibody titers and cross-neutralizing activity would accelerate vaccine development; however, a lack of uniformity in target strains used by different investigators to assess cross-neutralization has made the comparison of vaccine-induced antibody responses difficult. Thus, there is an urgent need to establish standard panels of HIV-1 reference strains for wide distribution. To facilitate this, full-length gp160 genes were cloned from acute and early subtype B infections and characterized for use as reference reagents to assess neutralizing antibodies against clade B HIV-1. Individual gp160 clones were screened for infectivity as Env-pseudotyped viruses in a luciferase reporter gene assay in JC53-BL (TZM-bl) cells. Functional env clones were sequenced and their neutralization phenotypes characterized by using soluble CD4, monoclonal antibodies, and serum samples from infected individuals and noninfected recipients of a recombinant gp120 vaccine. Env clones from 12 R5 primary HIV-1 isolates were selected that were not unusually sensitive or resistant to neutralization and comprised a wide spectrum of genetic, antigenic, and geographic diversity. These reference reagents will facilitate proficiency testing and other validation efforts aimed at improving assay performance across laboratories and can be used for standardized assessments of vaccine-elicited neutralizing antibodies.

Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies

Globally, human immunodeficiency virus–type 1 (HIV-1) is extraordinarily variable, and this diversity poses a major obstacle to AIDS vaccine development. Currently, candidate vaccines are derived from isolates, with the hope that they will be sufficiently cross-reactive to protect against circulating viruses. This may be overly optimistic, however, given that HIV-1 envelope proteins can differ in more than 30% of their amino acids. To contend with the diversity, country-specific vaccines are being considered, but evolutionary relationships may be more useful than regional considerations. Consensus or ancestor sequences could be used in vaccine design to minimize the genetic differences between vaccine strains and contemporary isolates, effectively reducing the extent of diversity by half.

https://science.sciencemag.org/content/sci/296/5577/2354.full.pdf

Diversity considerations in HIV-1 vaccine selection.

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.

Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41.

The few antibodies that can potently neutralize human immunodeficiency virus type 1 (HIV-1) recognize the limited number of envelope glycoprotein epitopes exposed on infectious virions. These native envelope glycoprotein complexes comprise three gp120 subunits noncovalently and weakly associated with three gp41 moieties. The individual subunits induce neutralizing antibodies inefficiently but raise many nonneutralizing antibodies. Consequently, recombinant envelope glycoproteins do not elicit strong antiviral antibody responses, particularly against primary HIV-1 isolates. To try to develop recombinant proteins that are better antigenic mimics of the native envelope glycoprotein complex, we have introduced a disulfide bond between the C-terminal region of gp120 and the immunodominant segment of the gp41 ectodomain. The resulting gp140 protein is processed efficiently, producing a properly folded envelope glycoprotein complex. The association of gp120 with gp41 is now stabilized by the supplementary intermolecular disulfide bond, which forms with approximately 50% efficiency. The gp140 protein has antigenic properties which resemble those of the virion-associated complex. This type of gp140 protein may be worth evaluating for immunogenicity as a component of a multivalent HIV-1 vaccine.

A Recombinant Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Complex Stabilized by an Intermolecular Disulfide Bond between the gp120 and gp41 Subunits Is an Antigenic Mimic of the Trimeric Virion-Associated Structure

The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs’ abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events.

https://jvi.asm.org/content/jvi/73/5/4145.full.pdf

Differential Inhibition of Human Immunodeficiency Virus Type 1 Fusion, gp120 Binding, and CC-Chemokine Activity by Monoclonal Antibodies to CCR5

The native, fusion-competent form of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex is a trimeric structure composed of three gp120 subunits and three gp41 subunits; the receptor-binding (CD4 and coreceptor) sites are located in the gp120 moieties, and the fusion peptides are located in the gp41 components (10, 33, 34, 52, 69, 78, 83). In the generally accepted model of HIV-1 fusion, the sequential binding of gp120 to CD4 and a coreceptor induces a series of conformational changes in the gp41 subunits, leading to the insertion of the fusion peptides into the host cell membrane in a highly dynamic process (14, 31, 39, 59, 68, 72, 81, 84, 91). The associations between the six components of the fusion-competent complex are maintained via noncovalent interactions between gp120 and gp41 and between the gp41 subunits (52, 84). These interactions are relatively weak, making the fusion-competent complex unstable. This instability perhaps facilitates the conformational changes in the various components that are necessary for the fusion reaction to proceed efficiently, but it greatly complicates the task of isolating the native complex in purified form. Put simply, the native complex falls apart before it can be purified, leaving only the dissociated subunits.

One reason it would be desirable to produce the native HIV-1 envelope complex is to explore its potential as an immunogen, perhaps after modification to improve its exposure of critical neutralization epitopes. The limited neutralizing-antibody response to HIV-1 in infected people is directed at the native complex and is probably raised against it (6, 40, 49, 51); P. W. H. I. Parren, D. R. Burton, and Q. J. Sattentau, Letter, Nat. Med. 3:366, 1997). In contrast, the isolated subunits have not proven efficient at inducing relevant neutralizing antibodies (reviewed in references 6, 49, and 51). We and others are therefore attempting to make more-stable forms of the envelope glycoprotein complex that better mimic the native structure. Usually, these efforts have focused on making various forms of soluble gp140 glycoproteins which contain gp120 but only the ectodomain of gp41 (4, 11, 13, 17, 19-21, 57, 66, 76, 85-87, 90).

An approach to resolving the instability of the native complex is to remove the cleavage site that naturally exists between the gp120 and gp41 subunits. Doing so means that proteolysis of this site does not occur, leading to the expression of gp140 glycoproteins in which the gp120 subunit is covalently linked to the gp41 ectodomain (gp41ECTO) by means of a peptide bond (2, 3, 16-18). Such proteins can be oligomeric, sometimes trimeric (11, 16-21, 54, 66, 85-87, 90). However, it is not clear that they truly represent the structure of the native, fusion-competent complex in which the gp120-gp41 cleavage site is fully utilized. Hence the receptor-binding properties of uncleaved gp140 (gp140UNC) proteins tend to be impaired, and nonneutralizing antibody epitopes are exposed on them that probably are not accessible on the native structure (4, 6, 28, 60, 90).

We have taken an alternative approach to the problem of gp120-gp41 instability, which is to retain the cleavage site but to introduce a disulfide bond between the gp120 and gp41ECTO subunits (4, 57). Properly positioned, this intermolecular disulfide bond forms efficiently during envelope glycoprotein (Env) synthesis, allowing the secretion of gp140 proteins that are proteolytically processed but in which the association between the gp120 and gp41ECTO subunits is maintained by the disulfide bond.

Here we show that the gp41-gp41 interactions are unstable in the SOS gp140 protein, which is expressed and purified primarily as a monomer. In contrast, gp140UNC proteins—with or without the SOS cysteine substitutions—are multimeric, implying that cleavage of the peptide bond between gp120 and gp41 destabilizes the native complex. Despite being monomeric, the purified and unpurified forms of SOS gp140 are better antigenic structural mimics of the native, fusion-competent Env structure than are the corresponding gp120 or gp140UNC proteins. This may be because the presence and orientation of gp41ECTO occludes certain nonneutralization epitopes on SOS gp140 while preserving the presentation of important neutralization sites. This explanation is consistent with immunoelectron microscopy studies of the protein. Unexpectedly, proteolytically mature, but variable-loop-deleted, SOS gp140 glycoproteins have enhanced oligomeric stability, so these molecules warrant further study for their structural and immunogenic properties.

Oligomeric and Conformational Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140 Envelope Glycoprotein

Deborah J. Anselma

Papers

A Recombinant Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Complex Stabilized by an Intermolecular Disulfide Bond between the gp120 and gp41 Subunits Is an Antigenic Mimic of the Trimeric Virion-Associated Structure

Differential Inhibition of Human Immunodeficiency Virus Type 1 Fusion, gp120 Binding, and CC-Chemokine Activity by Monoclonal Antibodies to CCR5

Oligomeric and Conformational Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140 Envelope Glycoprotein

by Monoclonal Antibodies to CCR5 gp120 Binding, and CC-Chemokine Activity Immunodeficiency Virus Type 1 Fusion, Differential Inhibition of Human