An effective microbicide must have (1) a potent and preferentially broad-spectrum antimicrobicidal activity, (2) limited to no toxicity on the vaginal or rectal epithelial cell layer, and (3) a good pharmacokinetic/dynamic profile.29
Disruption of the epithelial layer increases the risk for sexually transmitted diseases such as HIV and herpes simplex virus type 2 (HSV-2).30
From all the diverse CBAs described so far, GRFT is presumably the most potent and broad-spectrum anti-HIV-1 entry inhibitor with an outstanding safety and efficacy profile.6,8
Recombinant forms of GRFT, with no loss of antiviral activity, can also be easily produced in E. coli
and in the Nicotiana benthamiana
Also BanLec ranks among the most potent anti-HIV-1 lectins reported so far.5
Although GRFT and BanLec have been reported to suppress a wide variety of HIV-1 strains and clinical isolates, the HIV-2 inhibitory activity of GRFT and BanLec has not previously been reported. When the efficacy of GRFT was evaluated against HIV-2 (ROD) replication, a range of EC50
values between 110 and 240
pM was obtained (). These values of antiviral activity were only 2- to 3-fold higher than those observed for inhibition of HIV-1 (NL4.3), since the EC50
s for HIV-1 (NL4.3) varied between 48 and 75
pM (). When the anti-HIV-2 activity of BanLec was compared with HIV-1, no significant differences in inhibitory concentrations were observed (EC50
nM for HIV-1 and 0.13±0.01
nM for HIV-2; and ). Thus, both GRFT and BanLec efficiently suppress both HIV-1 and HIV-2 in the picomolar range.
In the search for an effective, safe, acceptable, and affordable microbicide, many potential microbicidal candidates were tested alone or in combination with other (classes of
) antiretroviral drugs.31,32
However, no investigations were performed on the effects on HIV replication when two drugs belonging to the functional class of CBAs would be combined. To evaluate synergistic drug–drug interactions, some research groups propose to use “single-round” instead of “multiple-round” infection assays to evaluate synergy.33–35
Recently a paper by Ketas et al.
was published that suggested a modification of the classic synergy calculation by the Chou and Talalay method as they used single-round infection and a nonlinear calculation model.33
Here, we used the multiple-round virus infection to mimic more the real viral life cycle and replication abilities in a CD4+
T cell line and PBMCs.
It was interesting to observe synergistic anti-HIV activity when GRFT was combined with any other CBA, irrespective of the coreceptor tropism of the virus (X4 or R5) or the cell type (MT-4, PBMC). Such synergistic potential of GRFT was also kept against mutant HIV-1 strains that were (partially) resistant to other CBAs. BanLec in combination with GRFT (or MVN) also resulted in synergy. Although BanLec and GRFT lost some of their antiviral activity (~10-fold) against the HIV-1 NL4.32G12res and NL4.3MVNres strains compared to the original wild-type HIV-1 NL4.3 virus ( and ), they proved to be so exquisitely potent that they kept pronounced antiviral activity against the drug-resistant virus strains and, when combined, synergy is still observed. Based on these data, GRFT as well as BanLec or any nonmitogenic derivative thereof could be an interesting lead molecule for further combined microbicidal development.
CV-N and MVN are both CBAs isolated from Cyanobacteria and have a similar gp120 sugar binding specificity, directed against Manα(1,2)Man residues. CV-N has both potent anti-HIV-1 and anti-HIV-2 activity, but also strong cytokine-stimulatory and mitogenic activity.36,37
On the other hand, MVN has comparable anti-HIV-1 activity to CV-N but a much higher safety profile.9,38
Therefore we used MVN instead of CV-N for exploring dual CBA combinations. In all, except one (GRFT/HHA), dual MVN/CBA combinations, synergistic activity was observed ( and ). The additivity between GRFT and HHA can possibly be explained by the sharing of a similar epitope on gp120 by both CBAs.
Strong synergism was observed between GRFT and many other lectin CBAs as discussed above but also when combined with the carbohydrate-binding mAb 2G12 (CI, 0.29±0.04), although 2G12 mAb has a much lower antiviral activity as a single agent compared to GRFT. Remarkably, while rather low-dose reductions were observed in most of the evaluated dual CBA combinations, the 2G12 mAb had a 9-fold dose reduction in MT-4 cells and even up to 13-fold in PBMCs, which could have potential benefits when it would be used in a combined microbicidal gel application. These synergy data are in agreement with recently published data on pseudo-typed HIV-1 using MVN and CV-N in combination with 2G12 mAb.38
As also observed in , the broadly neutralizing 2G12 mAb did not completely block viral replication even at high doses and thus may lead to the rapid appearance of resistant virus. In fact, deletion of one specific glycan mutation at position 295 (N295) in gp120 proved sufficient to display full resistance to 2G12 mAb.25
Also, it has been demonstrated that the binding of 2G12 mAb to gp120 can be blocked by MVN and BanLec but not vice versa as 2G12 mAb does not block the binding of MVN or BanLec to gp120.5,9
These findings can be explained by the assumption that the lectin CBAs can bind to several sugars on gp120, whereas 2G12 mAb recognizes only one well-defined sugar epitope. The incomplete suppression of virus replication by 2G12 mAb and the subsequent risk of resistance development are other arguments for combining 2G12 mAb with a lectin CBA. Our data revealed not only full virus suppression in such CBA/2G12 mAb drug combination experiments, but also a pronounced synergistic antiviral activity. The observed synergy can have multiple explanations, i.e., targeting of multiple but distinct gp120 (N-glycan) epitopes by different CBAs, different time points of CBA interaction during the gp120/gp41-CD4/CXCR4/CCR5 binding and fusion process, and/or conformational changes in gp120 upon binding of one CBA causing exposure of previously less available epitopes allowing a more efficient binding by the other CBA. The synergy/additivity between the CBAs can also be explained by subtle differences in the CBA sugar specificity.
With the non-CBA mAb b12, targeting the CD4 binding site on gp120, synergism occurred in combination with MVN and GRFT (). Alexandre et al.
showed that GRFT interacts, among others, with the N-glycan at position 386 (N386) on gp120, and exposes the CD4 binding site by binding to this glycan. This allows a more tight interaction of the GRFT-bound gp120 with the mAb b12, which may explain the synergistic activity.39
It could therefore be possible that a similar mechanism of action is responsible for the observed synergy between MVN and the non-CBA mAb b12. Our generated HIV-1 NL4.3MVNres
with a deleted N-linked glycan on position 386 indeed demonstrated that N386 is a crucial anchoring point for MVN.9
For actinohivin, a broadly neutralizing prokaryotic lectin, a tight interaction with the envelope protein of SIV was demonstrated by surface plasmon resonance technology, but it lacked anti-SIV activity in several viral replication assays.40
These data revealed that binding of CBAs to gp120 glycans may occur, but these are not always necessarily in neutralizing viral replication. Based on these findings, we investigated whether the CBAs 2G12 mAb and MVN, which both lack anti-HIV-2 activity, could influence the anti-HIV-2 activity of GRFT by binding to gp120 and triggering conformational changes in the envelope that may influence GRFT binding and eventual antiviral efficacy or vice versa. However, no increase or decrease in the anti-HIV-2 effect of GRFT was seen in the paired drug combinations (). Neither did binding of GRFT to gp120 induce conformational changes throughout gp120 that led to a gain of anti-HIV-2 activity for MVN or 2G12 mAb.
In contrast to most other paired CBA combinations, no synergy was observed between HHA and GNA against the wild-type HIV-1 strains NL4.3 (X4) and BaL (R5) and against HIV-2 ROD (– and ). Instead, against the NL4.32G12res
HIV-1 strains, HHA/GNA clearly showed synergistic interactions ( and ). Both HHA and GNA are tetrameric plant lectins with a rather high molecular weight (50
kDa). These agents recognize structurally comparable N-glycans on gp120, namely α(1,3) and α(1,6)Mannose residues,3
which seems to result in an additive combinatory drug profile. Surprisingly, HIV-1 NL4.32G12res
virus was strikingly more susceptible to the CBAs HHA and GNA than wild-type virus.25
This peculiar phenomenon is again shown here when both CBAs were combined, resulting in a ~10-fold decrease of HHA concentration from 3.8
nM to 0.37
nM and a 35-fold decrease of GNA concentration from 15
nM to 0.43
nM ( and ). The deleted N-glycans in the mutant gp120 of the CBA-resistant virus strains may create “holes” in the N-glycan shield and/or conformational changes on the surface of gp120 resulting in a better “binding site” for certain high-molecular-weight CBAs such as HHA and GNA to allow synergy.
The microbicide 1% tenofovir gel study (CAPRISA 004) revealed a reduced transmission of HIV-1 but also of HSV-2 by 39% and 51%, respectively.41
The latest results of the VOICE (Vaginal and Oral Interventions to Control the Epidemic) study (MTN-003) were less optimistic. The oral tenofovir tablet and tenofovir gel arms of VOICE were dropped following interim reviews of data that determined neither product was effective in the women assigned to those study groups. Adherence seemed to be the major problem, although many other questions remain.
A recent study has shown that tenofovir inhibits HSV-2 infections by targeting the herpetic DNA polymerase,42
which was made possible by the high local tenofovir concentrations that were afforded by the topical drug delivery. A microbicide drug with such a dual mechanism of antiviral (i.e., HIV and HSV) action is beneficial in terms of clinical application since HSV-2 is considered as an important copathogen that may accelerate HIV transmission in HIV-exposed individuals. Interestingly, besides its potent anti-HIV activity, GRFT also inhibits HSV-2 (K.E. Palmer et al.,
unpublished observations). Since we could show that tenofovir acts synergistically with various CBAs including GRFT, MVN, and BanLec,43,44
a GRFT/tenofovir combination gel may become a very attracting microbicide formulation to diminish HIV-1 and HIV-2 but also HSV-2 transmission worldwide. Additionally, due to the potent anti-HCV activity of GRFT and other CBAs, such agents could even be able to reduce the number of novel (co)infections with HCV.45,46
Within the scope of the topical PrEP perspective, the CBAs have the benefit of their broad antiviral activity, and by strongly binding to the glycans on gp120 as a virucide they target HIV in the lumen of the vagina before genital tissue penetration can occur. In addition, they have also the advantage of a high genetic barrier to resistance and do not need cellular uptake as well as activation/modification to gain anti-HIV activity (e.g., the RTI tenofovir). Even the important DC-SIGN-mediated pathway (HIV capture and transmission to susceptible CD4+
T cells) can be completely blocked by this class of inhibitors.3,47
The first trials in macaques using gel-formulated CV-N applied vaginally and rectally showed promising results as it potently inhibited SHIV transmission.48,49
More recently, a 63% reduction in SHIV transmission was observed when macaques were treated with a CV-N-expressing Lactobacillus
Given the many failures in clinical trials of polyanionic antivirals (e.g., nonoxynol-9 and PRO2000),51,52
which also target HIV in the lumen of the vagina, the class of CBAs (especially GRFT) could contribute to novel perspectives in the field of PrEP.
In conclusion, our study has shown synergistic antiviral (HIV-1/HIV-2) activity when dual combinations of CBAs were exposed to virus-infected cell cultures, irrespective of the nature of the virus (X4, R5) and cell type (MT-4, PBMC). Also, virus strains containing N-glycan deletions in their envelope often maintain high sensitivity to the synergistic CBA combinations. These data are very encouraging in the search for the development of efficient drug pairs to be combined for microbicide treatment.