Neutralization of feline immunodeficiency virus by antibodies targeting the V 5 loop of Env

Received 27 July 2009 Accepted 17 September 2009 Neutralizing antibodies (NAbs) play a vital role in vaccine-induced protection against infection with feline immunodeficiency virus (FIV). However, little is known about the appropriate presentation of neutralization epitopes in order to induce NAbs effectively; the majority of the antibodies that are induced are directed against non-neutralizing epitopes. Here, we demonstrate that a subtype B strain of FIV, designated NG4, escapes autologous NAbs, but may be rendered neutralization-sensitive following the insertion of two amino acids, KT, at positions 556–557 in the fifth hypervariable (V5) loop of the envelope glycoprotein. Consistent with the contribution of this motif to virus neutralization, an additional three subtype B strains retaining both residues at the same position were also neutralized by the NG4 serum, and serum from an unrelated cat (TOT1) targeted the same sequence in V5. Moreover, when the V5 loop of subtype B isolate KNG2, an isolate that was moderately resistant to neutralization by NG4 serum, was mutated to incorporate the KT motif, the virus was rendered sensitive to neutralization. These data suggest that, even in a polyclonal serum derived from FIV-infected cats following natural infection, the primary determinant of virus-neutralizing activity may be represented by a single, dominant epitope in V5.


INTRODUCTION
Feline immunodeficiency virus (FIV) was first isolated in 1986 in Petaluma, CA, USA, from a cat with an immunodeficiency-like syndrome (Pedersen et al., 1987) and is an important pathogen of domestic cats worldwide.FIV is the feline homologue of human immunodeficiency virus (HIV) and these viruses share many biological and pathogenic features (Bendinelli et al., 1995;English et al., 1993;Johnson et al., 1994), typified by a selective targeting of CD4 + T lymphocytes and the induction of a progressive immunosuppression (Ackley et al., 1990).
The induction of an effective humoral response against lentivirus infection is a key element in the immunological control of the disease, and the primary target for neutralizing antibodies (NAbs) is the virus envelope glycoprotein (Env) (Binley et al., 2004;Frost et al., 2005;Wei et al., 2003).Env varies by up to 30 % amongst FIV subtypes (Hosie & Beatty, 2007) and, thus, the preparation of an immunogen capable of inducing broadly neutralizing antibody responses against such diverse isolates of FIV would be of great value to the development of an FIV vaccine.Further, the development of a vaccine against FIV would have implications extending beyond veterinary medicine; FIV is the only non-primate lentivirus that induces AIDS-like symptoms in its natural host and, as such, is a valuable animal model for both prophylactic and therapeutic studies for HIV (Bendinelli et al., 1995;Elder et al., 1998;Okada et al., 1994).Moreover, cats have the advantage of being easier to breed and have shorter life cycles than other animal models currently used for HIV research (Miller et al., 2000).
Previous studies showed that vaccination with whole inactivated vaccines or DNA vaccines can protect cats against challenge with either low-virulence/homologous strains of FIV, but not against virulent/heterologous primary strains (reviewed by Hosie & Beatty, 2007).A vaccine capable of protecting cats against infection with a wide range of virulent primary isolates remains a challenge, primarily because of the limited number of conserved neutralization epitopes that have been identified and the failure to present such epitopes appropriately in vaccines.Steric factors may render neutralization epitopes inaccessible to NAbs (Chiarantini et al., 1998;Labrijn et al., 2003), whilst most of the antibodies generated by vaccination may The GenBank/EMBL/DDBJ accession numbers for the novel FIV env sequences generated in this study are GU066862-GU066866.be directed against non-neutralizing epitopes (Dhillon et al., 2007;Richman et al., 2003).Accordingly, the identification of conserved determinants for neutralization will be an important advance in the design of the next generation of FIV vaccines.

Supplementary tables showing oligonucleotides
Understanding the biological basis for the induction of antibodies with broad neutralizing activity is pivotal to the development of efficacious lentivirus vaccines and to the generation of vaccines that will protect against a broad range of primary isolates.Encouraging results have indicated that it is possible to protect cats against challenge with primary FIV isolates, depending on their regional distribution.By vaccinating cats with a whole inactivated vaccine based on the subtype B Pisa M2 strain, cats were protected from natural challenge with subtype B isolates of FIV (Pistello et al., 1997).It has been suggested that subtype B isolates may be more ancient and, accordingly, more host-adapted (Bachmann et al., 1997), hence they may be more readily neutralizable by the host humoral response.In contrast, whilst subtype A viruses display similar levels of non-synonymous mutations, such viruses display half as many synonymous mutations, suggesting a more recent spread and a lower level of host-virus adaptation (Bachmann et al., 1997).As the major FIV subtypes are restricted in their global distribution, it has been proposed that it may be necessary to design several regional vaccines rather than a single, worldwide-protective vaccine (Steinrigl & Klein, 2003).
In this study, we investigate the initial observation that a subtype B strain resisted neutralization by its homologous serum.By applying targeted mutagenesis across the env gene, we identified the V5 loop as the primary determinant of escape from neutralization and localized the primary determinant to a lysine-threonine (KT) motif.Additional subtype B strains bearing the same motif were also sensitive to neutralization.Furthermore, incorporation of these residues into a neutralization-resistant subtype B strain rendered the virus more sensitive to neutralization, confirming the monospecificity of the neutralizing serum.These data indicate that the functional neutralizing response in natural infection with FIV may be highly specific and that immune evasion in vivo may stem from the mutation of as few as two amino acids in the V5 loop.

Phylogenetic analysis
An unrooted neighbour-joining phylogenetic tree was generated based on 651 nt spanning the env V3-V5 sequence from the five isolates included in the study, as well as reference sequences from subtypes A, B, C, D and E. The phylogenetic tree (Fig. 1) demonstrates that the five Japanese isolates were genetically related and clustered within subtype B with bootstrapping support of 100 %.KNG1 and TOT1 were highly similar at the nucleotide level and indeed had identical V3-V5 amino acid sequences and clustered together on a single tree branch (Fig. 1).

Cats show different NAb responses to homologous and heterologous strains
Sera from cats KNG1 and TOI1 showed no evidence of neutralizing activity against any of the pseudotypes tested, including those bearing their homologous Env proteins, and serum from cat KNG2 showed no neutralizing activity against the KNG1, NG4 or TOT1 pseudotypes, moderate neutralizing activity against the KNG2 pseudotype and weak neutralizing activity against the TOI1 pseudotype (Table 1).In contrast, sera from cats NG4 and TOT1 showed strong neutralizing activity against pseudotypes expressing Env proteins of the heterologous KNG1, TOT1 and TOI1 strains, and moderate responses against pseudotypes expressing the Env of strain KNG2 .The pseudotype expressing the NG4 Env resisted neutralization by four heterologous sera and showed only weak neutralizing activity against its homologous serum (Table 1).Although KNG1 and TOT1 shared similar env sequences (97 % similarity), they did not induce similar humoral immune responses in their respective hosts.

Escape from homologous neutralization by NG4
NG4 was neutralized only weakly or resisted neutralization by the two cross-neutralizing sera NG4 and TOT1, respectively.The finding that NG4 was neutralized only weakly by its homologous serum, a serum that crossneutralized the other four viruses, may indicate the evolution in vivo of a neutralization-escape mutant that, at the time of sampling, was the dominant species in the periphery, as five independent Env clones for each virus were sequenced, all of which gave rise to identical V5 amino acid sequences (data not shown).However, as blood samples from cat NG4 at earlier time points in infection were not available, it was not possible to identify earlier strains that were most likely to have contributed to the generation of the NAbs.However, given the similarity between the five subtype B isolates, sites of amino acid divergence from the consensus sequence that were identified in NG4 would be likely to contribute to the relative resistance of this strain to neutralization by its homologous serum.Accordingly, these non-synonymous residues were targeted by site-directed mutagenesis in an attempt to render NG4 more sensitive to neutralization by its homologous serum.Initially, Env was mutated at the V3 loop, a known target for NAbs (Lombardi et al., 1993;Osborne et al., 1994).A glutamine residue (377) was substituted with a histidine (Q377H), resulting in a net increase in the charge of the V3 loop.However, the Q377H mutation did not render NG4 more sensitive to neutralization (Fig. 2).Next, the NG4 Env was mutated at the crown of the predicted V5 loop (Fig. 3), targeting serine residue 550 (S550N), creating a potential site for N-linked glycosylation.When tested against its homologous serum, the S550N mutant did not show enhanced sensitivity to neutralization (Fig. 2; wild type, 46 % neutralization at a 1 : 10 dilution; S550N, 38 % at a 1 : 10 dilution), indicating that the absence of asparagine 550 alone and ablation of the predicted site for N-linked glycosylation did not contribute to the escape from neutralization by homologous serum.The NG4 Env was then mutated within the V5 loop by the insertion of two amino acids, lysine and threonine, at positions 556 and 557 [S556(KT)557K].Interestingly, this mutation rendered NG4 sensitive to neutralization, with strong neutralizing activity (98 %) observed at a 1 : 10 dilution (Fig. 2a, b).Moreover, the S556(KT)557K insertion also rendered NG4 sensitive to the broadly neutralizing TOT1 serum (Fig. 2c, d), indicating that, despite the independent origins of the two viruses, both cats responded to infection by targeting V5.Next, we asked whether the increase in polarity afforded by the inserted lysine residue contributed to the formation of the neutralizing determinant.An S556(K)557K mutant was prepared in which only a lysine residue was inserted.The NG4 S556(K)557K mutant was tested against NG4 serum and showed an intermediate pattern of neutralization (Fig. 2), indicating that, whilst the lysine residue was clearly important, the additional threonine residue enhanced the formation of the neutralizing determinant.These data emphasize the dominance of V5 in the neutralization of FIV and are consistent with a hypothesis whereby the broadly neutralizing NG4 serum selected for mutant viruses bearing a deletion in V5 in the host animal.

Neutralization sensitivity of KNG2
Of the five isolates studied, KNG2 was the most divergent at V5.In comparison with the other strains, KNG2 was moderately sensitive to neutralization by all sera tested, including the NG4 and TOT1 sera that showed strong neutralizing activity against KNG1, TOT1 and TOI1.Based on our finding that NG4 could be rendered sensitive to neutralization by a single change in the amino acid sequence of the V5 loop, we asked whether the partial resistance of KNG2 was conferred by its divergent V5 loop.Firstly, a KNG2 mutant was prepared bearing a DN556KT mutation, targeting the same residues shown earlier to confer neutralization sensitivity upon NG4.The DN556KT mutation did not display improved sensitivity to neutralization relative to the wild type.As the sequence of KNG2 diverged from the consensus at 7 aa, we considered that the context of the mutation may have been important for formation of the epitope.Accordingly, the V5 loop was mutated in stages towards identity with the (highly sensitive) KNG1 and TOT1 sequences; initially a 5 aa change was created (553NGNDN558A553DNSDT558), followed by the full mutation (551STNGNDNKMT558A 551GTDNSKTKMA558).Both KNG2 mutants were then tested for sensitivity to neutralization by the TOT1 serum.The 551STNGNDNKMT558A551GTDNSKTKMA558 variant was neutralized efficiently by the TOT1 serum, whilst the 553NGNDN558A553DNSDT558 mutant showed an intermediate sensitivity (Fig. 4).The conversion of KNG2 from neutralization-resistant to neutralization-sensitive by changing the sequence of V5 to a sequence similar to those of the neutralization-sensitive KNG1 and TOT1 strains is consistent with V5 being a primary determinant of neutralization sensitivity and the specificity of the neutralizing sera.
The results of this study indicate the importance of V5 to antibody-mediated neutralization of FIV.Whilst anti-V5 antibodies can neutralize primary isolates of FIV very efficiently, the virus appears able to escape neutralization readily by mutating V5.What, then, are the prospects for designing an FIV vaccine that induces broadly neutralizing antibodies targeting V5?A comparison of published V5 sequences (Fig. 5) would suggest that the V5 region has highly conserved structural features, such as the cysteines at positions 533, 546, 548 and 561 (numbering as in KNG1 and 2, TOI1 and TOT1; Fig. 5).However, the region between cysteine residues 548 and 561 appears to be remarkably flexible in its ability to tolerate amino acid substitutions, glycosylation (potentially O-and N-linked) and length polymorphisms, suggesting that it is under pressure to vary from the host immune response.The relative conservation of the region between cysteines 533 and 548 may indicate that this region is either inaccessible or structurally constrained, perhaps through interactions with adjacent regions of Env.If possible, targeting the humoral response to this region may induce more broadly neutralizing antibodies.

DISCUSSION
In this study, we demonstrate that the V5 loop of FIV Env is a primary target for virus-neutralizing antibodies.
Although previous studies in vitro indicated that the V3 loop was a major determinant for virus neutralization, subsequent studies revealed that the significance of antibodies targeting this region to the neutralizing response had been overemphasized by the cellular substrate used to quantify neutralization.Formerly, assays were based on inhibiting infection of Crandell feline kidney (CrFK) cells with 'CrFK-adapted' strains of virus.Prior to measuring neutralization in this system, the challenge virus had to be selected for growth in CrFK cells, thereby selecting for viruses that were capable of CD134-independent infection through a direct interaction with CXCR4 (analogous to CD4-independent infection with HIV).The V3 loop plays a critical role in the FIV Env-CXCR4 interaction; accordingly, CrFK-based assays exaggerated the importance of NAbs binding this region.Subsequent studies revealed that, when primary (non-CrFK-adapted) isolates were assayed on interleukin-2-dependent T cells, the correlation between the efficiency of virus neutralization and the response to V3 was diminished.Here, we extend previous studies that indicated that, in biologically relevant in vitro systems, the variable loops V4 and V5 appear to be the major targets for effective NAbs.Moreover, we found that, in polyclonal sera from infected cats, deletion of as few as two amino acids from V5 converts a virus from a strongly neutralized to a weakly neutralized or neutralization-resistant phenotype.Each of the isolates described in the current study was confirmed as being CD134dependent by plating the virus pseudotypes on target cells expressing CD134.No evidence of CD134-independent infection via CXCR4 alone was detected (data not shown), confirming that the isolates were primary and not cellculture-adapted.
Amongst the variable regions in FIV Env, V5 appears to be highly polymorphic between strains, with variations in amino acid sequence, glycosylation and extended stretches of serine and threonine residues being common.These data suggest that there is a selective pressure within the host for variation in V5, consistent with escape from an effective immune response.The fact that minimal variation in V5 is sufficient to either reduce substantially or ablate completely the neutralizing capacity of the host humoral response is of concern for the design of effective FIV vaccines.How, then, are we to elicit broadly neutralizing antibodies in vivo?
Clearly, the V4 and V5 loops are exposed and immunogenic, and antibodies targeting these regions neutralize virus.However, it would appear that these regions may represent little more than a decoy for the virus, presenting the immune system with an easy target for NAbs.By the time functional NAbs develop, virus variants that can escape the monospecific serum may have already evolved in vivo.The solution to this dilemma would appear to lie in the identification of framework determinants in Env that induce antibodies that are broadly active, refocusing the immune response either by silencing the immunodominant (but ultimately ineffectual) determinants in V4 and V5, or by presenting them in a such a way as to induce antibodies that recognize structures or motifs that are difficult or impossible for the virus to vary.
Previous studies have demonstrated the importance of V5 in virus neutralization (Giannecchini et al., 2001;Pistello et al., 2003;Siebelink et al., 1995).V5 was mapped by using synthetic peptides for a possible linear epitope based on the sequence of the 19k1-560 strain (Siebelink et al., 1995), and it was demonstrated that a single mutation at position 560 may be involved concurrently with another mutation at 483 (V4 loop) in one or more conformational epitopes, but no linear epitope was mapped in V5.Further, two independent mutations have been reported that resulted in the creation of potential N-linked glycosylation sites in V4 (K481N) and V5 (S557N) and which contributed to the conversion from a neutralization-sensitive to a neutralization-resistant phenotype (Giannecchini et al., 2001;Pistello et al., 2003).In comparison, the conversion from a weakly neutralized or neutralization-resistant to a strongly neutralized phenotype described herein was mediated by mutation of V5 alone.The V5 loop of FIV Env is highly variable among isolates.Variation occurs not only within the amino acid sequence, but also in the length of the central region (up to 14 residues) towards the end of the loop formed by disulfide-bond linkage, indicating that this particular region may play a significant role in virus evolution and escape from neutralization.It is notable that the length polymorphisms in this region are attributable to a reiteration of codons encoding serine and threonine residues.Threonine and serine have hydroxyl side chains and may, potentially, undergo O-linked glycosylation.It is generally believed that, due to their small molecular size, O-linked oligosaccharides have only minor effects on the formation of the glycan shield on HIV Env compared with N-linked oligosaccharides.However, these oligosaccharides usually have diverse structures with no common carbohydrate core and it is difficult to predict how efficient they may be in shielding neutralization epitopes, especially when there are several adjacent molecules (Bernstein et al., 1994).Moreover, X-ray crystallography studies on HIV-1 gp120 showed that glycosylation may have significant effects on the conformational stability of Env, as well as an indirect effect on more distant sequences along the secondary structure.This, in turn, would affect the interaction between the epitope and NAbs, rendering epitopes less accessible for antibody binding (Huang et al., 1997).
In conclusion, we identified a neutralization determinant in the FIV V5 loop that is associated with the conversion of strongly neutralized to weakly neutralized or neutralization-resistant strains.The determinant is apparently linear and involves at least 10 aa (GTDNSKTKMA) within the V5 region, a highly variable region amongst FIV strains.The identification of this neutralization determinant will inform vaccine design in the future in order to induce more potent NAb responses.

METHODS
Blood samples and collection of peripheral blood mononuclear cells (PBMCs) and sera.All blood samples were collected from five naturally infected cats from three different regions of Japan (isolate NG4 originated in Isikawa, KNG1 and KNG2 from Kanagawa and TOI1 and TOT1 from Tokyo).Four cats (KNG1, NG4, TOT1 and TOI1) were apparently healthy and displayed no clinical signs (analogous to the asymptomatic stage of HIV infection), whereas one cat, KNG2, presented with severe gingivostomatitis (analogous to the symptomatic stage of HIV infection).PBMCs were fractionated from 5 ml heparinized whole blood by centrifugation over Ficoll-Paque density separation medium (GE Healthcare).Aliquots of sera were stored at 280 uC prior to use in neutralization assays.
Cloning and pseudotype virus production.DNA was extracted from PBMCs by using a DNA Whole Blood extraction kit (Qiagen).The FIV env gene was amplified by PCR, using forward and reverse primers containing recognition sites for the restriction enzymes SalI and NotI, and then cloned into a low-copy-number eukaryotic expression vector, VR1012 (VICAL Inc.).To prepare HIV(FIV) pseudotypes, 3.3610 5 HEK-293T cells were transfected with 5 mg VR1012 expressing the FIV env clone and 5 mg pNL-Luc-E-R+ (Connor et al., 1995) (an Env-deleted HIV provirus that incorporates a luciferase reporter gene) by using SuperFect reagent (Qiagen), following the manufacturer's instructions.Culture supernatants were harvested at 48-72 h post-transfection, filtered at 0.45 mm and frozen at 280 uC until required.
DNA sequencing.The FIV env genes were sequenced by using a BigDye Terminator v1.1 kit (Applied Biosystems).The reaction mixture consisted of 100 ng purified DNA ml 21 , 3.2 pmol each primer, 4 ml sequencing buffer and 2 ml sequencing enzyme in 20 ml reactions.Cycling conditions were one cycle at 96 uC for 1 min, followed by 25 cycles at 96 uC for 10 s, 50 uC for 5 s and 60 uC for 4 min.Sequencing was performed by using an ABI3700 automated capillary array sequencer (Applied Biosystems).Raw chromatographic data were analysed by using 'Contig Express' sequenceanalysis software within the Vector NTI suite of programs (Invitrogen).
used for site-directed mutagenesis of V5 and GenBank numbers for new and reference sequences are available with the online version of this paper.Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Wed, 05 Dec 2018 23:25:41

Fig. 1 .
Fig. 1.Midpoint-rooted neighbour-joining tree of the FIV V3-V5 env gene sequence.The tree shows the five Japanese sequences included in the study (in bold type), as well as reference sequences from subtypes A, B, C, D and E.

Fig. 3 .
Fig. 3. Schematic structural model of the FIV SU protein, illustrating the locations of the Q377H mutation in V3 and of the V5 loop.The inset displays a comparison of the sequences from the five study strains, KNG1, KNG2, TOI1, TOT1 and NG4, and the reference strain TM2 (GenBank accession no.M59418).

Fig. 5 .
Fig.5.Amino acid alignment of the V5 region from diverse FIVs.Within the framework of the predicted loop bounded by cysteines 533 and 561 (numbering as in KNG1 and 2, TOI1 and TOT1), extensive variation is evident, the majority of which resides between cysteines 548 and 561.Hypervariable residues are marked (red asterisks).Colours indicate side chains: red, acidic; white, hydrophobic; light blue, amido; orange, aromatic; dark blue, basic; pink, hydroxyl; green, proline; yellow, thiol.