In Vitro Anti-HIV-1 Activity of Chitosan Oligomers N-Conjugated with Asparagine and Glutamine

Chitosan oligomers (COS) are polysaccharides obtained by the hydrolyzation of chitosan. They are water-soluble, biodegradable, and have a wide range of beneficial properties for human health. Studies have shown that COS and its derivatives possess antitumor, antibacterial, antifungal, and antiviral activities. The goal of the current study was to investigate the anti-human immunodeficiency virus-1 (HIV-1) potential of amino acid-conjugated COS compared to COS itself. The HIV-1 inhibitory effects of asparagine-conjugated (COS-N) and glutamine-conjugated (COS-Q) COS were evaluated by their ability to protect C8166 CD4+ human T cell lines from HIV-1 infection and infection-mediated death. The results show that the presence of COS-N and COS-Q was able to prevent cells from HIV-1-induced lysis. Additionally, p24 viral protein production was observed to be suppressed in COS conjugate-treated cells compared to COS-treated and untreated groups. However, the protective effect of COS conjugates diminished by delayed treatment indicated an early stage inhibitory effect. COS-N and COS-Q did not show any inhibitory effect on the activities of HIV-1 reverse transcriptase and protease enzyme. The results suggest that COS-N and COS-Q possess an HIV-1 entry inhibition activity compared to COS and further studies to develop different peptide and amino acid conjugates containing N and Q amino acids might yield more effective compounds to battle HIV-1 infection.


Introduction
Natural polysaccharides are the most abundant natural polymers composed of sugar monomers linked to each other with glycosidic bonds. Up to date studies showed that polysaccharides possess health beneficial effects, not only in terms of nutrition but also as therapeutic agents. This led to the ever increasing attention focusing on polysaccharides to develop pharmaceuticals of novel natural origin. Starting with the first reported antiviral polysaccharides against mumps and influenza [1,2], several other reports have suggested that polysaccharides and their derivatives are potent antivirals [3]. The inhibition of human immunodeficiency virus 1 (HIV-1) by polysaccharides and their derivatives was also one of the reported antiviral activities [4][5][6]. Studies have also shown that sulfated polysaccharides from different sources showed beneficial effects against HIV infection either inhibiting cell entry or viral replication [7]. Additionally, reports of polysaccharide derivatization showed that inactive polysaccharides could present antiviral properties in vitro following the addition of different side chains to sugar monomers [8].
In this context, chitin is a polymer found abundantly in nature formed by N-acetylglucosamine units. It is arguably one of the most abundant polysaccharides after cellulose. It can be found in a broad range of organisms and in various tissues ranging from the

Inhibition of Syncytia Formation and HIV-Induced Lytic Effects
Primarily, asparagine-conjugated COS (COS-N) and glutamine-conjugated (COS-Q) were evaluated for their anti-HIV-1 properties by their potential to inhibi cytia formation on C8166 human T cells infected with syncytia inducing X4 tropic H strain (HIV-1RF). Microscopic images of HIV-1RF-induced syncytia inhibitory activ compounds are shown in Figure 1a. Syncytia formation on C8166 cells was observe recorded at day 2 post-infection. Cells infected with HIV-1 produce a high amou membrane proteins specific to HIV-1 binding which then interact with other infected and fuse with each other, resulting in a giant cell formation called syncytia [30]. Acco to the images of cells and the quantification of formed syncytia, COS-N and COS-Q both able to reduce the HIV-1-induced syncytia formation. Counting syncytia forma revealed that the COS-N-treated cells exhibited a smaller syncytia count than those tr with COS-Q (Figure 1b). Both COS-Q-and COS-N-treated cells exhibited less syn formation compared to COS-treated cells.  The conjugated COS were examined for their ability to protect C8166 cells from HIV-1-induced lysis. The formation of syncytia is followed by cell killing due to the bursting of multinucleated giant cells. The effects were analyzed by cell viability five days after infection. The results show that untreated cells showed 17.24% viability at day 5 post-infection compared to the untreated uninfected group. Treatment with COS-N and COS-Q dose-dependently decreased the HIV-induced cell lysis shown as increased viability (Figure 1c). At the concentration of 100 µg/mL, the COS-N-treated group showed 64.24% viability. This number was 58.21% for the COS-Q-treated cells. Dextran sulfate (DS) was used as a positive control (100 µg/mL) and showed a similar effect to COS-N, although higher with 78.24% viability. On the other hand, COS-treated cells showed 25.43% viability.

Inhibition of the HIV-1 p24 Antigen
In order to further confirm the anti-HIV-1 effect of the COS-N and COS-Q, the production of the p24 antigen, one of the main viral markers of HIV-1 infection, was measured by ELISA and Western blot. The p24 protein is a crucial viral marker usually tested to diagnose HIV-1 infection along with HIV-1 RNA and antibodies. It is the capsid protein that makes up most of the protein load of the HIV-1 virus. In this experiment, H9 cells were used due to this cell line being permissive for HIV-1 replication for longer periods compared to the other cell lines used in this assay. As the aim of current investigation was to detect p24 antigens, H9 cells were infected with the HIV-1 IIIB strain instead of syncytia-inducing HIV-1 RF and C8166 cells. The results of p24 antigen capture ELISA show that HIV-1-infected H9 cells expressed a significant amount of p24 antigen (Figure 2a The conjugated COS were examined for their ability to protect C8166 cells from HIV-1-induced lysis. The formation of syncytia is followed by cell killing due to the bursting of multinucleated giant cells. The effects were analyzed by cell viability five days after infection. The results show that untreated cells showed 17.24% viability at day 5 postinfection compared to the untreated uninfected group. Treatment with COS-N and COS-Q dose-dependently decreased the HIV-induced cell lysis shown as increased viability (Figure 1c). At the concentration of 100 μg/mL, the COS-N-treated group showed 64.24% viability. This number was 58.21% for the COS-Q-treated cells. Dextran sulfate (DS) was used as a positive control (100 μg/mL) and showed a similar effect to COS-N, although higher with 78.24% viability. On the other hand, COS-treated cells showed 25.43% viability.

Inhibition of the HIV-1 p24 Antigen
In order to further confirm the anti-HIV-1 effect of the COS-N and COS-Q, the production of the p24 antigen, one of the main viral markers of HIV-1 infection, was measured by ELISA and Western blot. The p24 protein is a crucial viral marker usually tested to diagnose HIV-1 infection along with HIV-1 RNA and antibodies. It is the capsid protein that makes up most of the protein load of the HIV-1 virus. In this experiment, H9 cells were used due to this cell line being permissive for HIV-1 replication for longer periods compared to the other cell lines used in this assay. As the aim of current investigation was to detect p24 antigens, H9 cells were infected with the HIV-1IIIB strain instead of syncytiainducing HIV-1RF and C8166 cells. The results of p24 antigen capture ELISA show that HIV-1-infected H9 cells expressed a significant amount of p24 antigen ( Figure 2a   This was in parallel to the Western blot results where the cellular p24 levels and p24 levels of supernatant were dose-dependently decreased by COS-N and COS-Q treatment ( Figure 2b). This suggested that the treatment with COS conjugates decreased the viral production of HIV-1 proteins.

Inhibition of HIV-1 Reverse Transcriptase (RT) and Protease Activity
To provide insights into the action mechanism of the COS-N and COS-Q anti-HIV effect, their ability to inhibit the enzymatic activities of HIV-1 RT and protease was investigated. Previous results indicating that COS conjugates were able to decrease HIV-1 reproduction, however, are not indicative of whether COS conjugates intervened in the step of the HIV-1 viral cycle. Some of the main drugs on the market such as azidothymidine and saquinavir are enzyme inhibitors. These inhibit HIV-1 RT to stop viral gene insertion or HIV-1 protease to stop HIV-1 protein maturation that forms new infectious virions [31,32]. Several natural products, including polysaccharides and their derivatives, were reported to inhibit one of the enzymes to exert their anti-HIV-1 activity [7,33,34]. Therefore, to elucidate the action mechanism of COS conjugate-mediated HIV-1 inhibition, initially, HIV-1 RT and protease inhibitory effects of COS-N and COS-Q were evaluated.
The results show that although COS-N and COS-Q both inhibited the HIV-1 RT to some extent at 100 µg/mL (23.32% for COS-N and 20.14% for COS-Q), it was not comparable to that of the azidothymidine (5 µM) treatment which almost completely inhibited the HIV-1 RT (97.81%) ( Figure 3a). COS treatment did not show any effect on HIV-1 RT activity. Similar results were obtained with the protease activity assay ( Figure 3b). The COS-Q and COS treatments were not inhibitory to HIV-1 protease. COS-N treatment inhibited 18.30% of the protease activity of the untreated control, whereas treatment with saquinavir (5 µM) almost completely inhibited the HIV-1 protease (98.61%). These results suggest that COS conjugates are not inhibitors of HIV-1 enzymes. Studies have shown that some other anti-HIV-1 compounds with amide derivatization acted as entry inhibitors of human viruses including HIV-1 blocking the virus-cell or cell-cell connections to inhibit the entry of the virus into the host cell [35][36][37]. Some of these compounds were only able to inhibit HIV-infection at the early stages and did not show any effect in later stages. Expectedly they did not show any inhibitory effect on HIV-1 RT, protease, and integrase. According to the obtained data, and the reports of entry inhibitors with amide derivatization, it was hypothesized that COS conjugates might be effective at the early stages of HIV-1 infection.

Inhibition of HIV-1 Reverse Transcriptase (RT) and Protease Activity
To provide insights into the action mechanism of the COS-N and COS-Q anti-HIV effect, their ability to inhibit the enzymatic activities of HIV-1 RT and protease was investigated. Previous results indicating that COS conjugates were able to decrease HIV-1 reproduction, however, are not indicative of whether COS conjugates intervened in the step of the HIV-1 viral cycle. Some of the main drugs on the market such as azidothymidine and saquinavir are enzyme inhibitors. These inhibit HIV-1 RT to stop viral gene insertion or HIV-1 protease to stop HIV-1 protein maturation that forms new infectious virions [31,32]. Several natural products, including polysaccharides and their derivatives, were reported to inhibit one of the enzymes to exert their anti-HIV-1 activity [7,33,34]. Therefore, to elucidate the action mechanism of COS conjugate-mediated HIV-1 inhibition, initially, HIV-1 RT and protease inhibitory effects of COS-N and COS-Q were evaluated.
The results show that although COS-N and COS-Q both inhibited the HIV-1 RT to some extent at 100 μg/mL (23.32% for COS-N and 20.14% for COS-Q), it was not comparable to that of the azidothymidine (5 μM) treatment which almost completely inhibited the HIV-1 RT (97.81%) (Figure 3a). COS treatment did not show any effect on HIV-1 RT activity. Similar results were obtained with the protease activity assay (Figure 3b). The COS-Q and COS treatments were not inhibitory to HIV-1 protease. COS-N treatment inhibited 18.30% of the protease activity of the untreated control, whereas treatment with saquinavir (5 μM) almost completely inhibited the HIV-1 protease (98.61%). These results suggest that COS conjugates are not inhibitors of HIV-1 enzymes. Studies have shown that some other anti-HIV-1 compounds with amide derivatization acted as entry inhibitors of human viruses including HIV-1 blocking the virus-cell or cell-cell connections to inhibit the entry of the virus into the host cell [35][36][37]. Some of these compounds were only able to inhibit HIV-infection at the early stages and did not show any effect in later stages. Expectedly they did not show any inhibitory effect on HIV-1 RT, protease, and integrase. According to the obtained data, and the reports of entry inhibitors with amide derivatization, it was hypothesized that COS conjugates might be effective at the early stages of HIV-1 infection.

Inhibition of HIV-1 Entry
To further understand the anti-HIV-1 mechanism of COS conjugates, a delayed addition experiment was carried out. The delayed addition of COS conjugates to the infected cells was expected to provide insights into which stages of HIV-1 infection they might intervene in. The results show that adding COS conjugates to the cell culture resulted in a

Inhibition of HIV-1 Entry
To further understand the anti-HIV-1 mechanism of COS conjugates, a delayed addition experiment was carried out. The delayed addition of COS conjugates to the infected cells was expected to provide insights into which stages of HIV-1 infection they might intervene in. The results show that adding COS conjugates to the cell culture resulted in a time-dependent decrease in cell viability (Figure 4a  HIV-1RF-infected C8166 cells were incubated for 0, 2 12 h before being treated with COS conjugates or AZT (azidothymidine, 5 μM). Syncytia form were counted at day 2 post-infection. (c) Uninfected C8166 cells were co-cultured with H9 c fected with HIV-1IIIB at a ratio of 10:1 and treated with COS conjugates (100 μg/mL) or AZT thymidine, 5 μM). Syncytia formation was counted at day 5 post-infection. Blank: Uninfec treated cells, Control: HIV-1-infected untreated cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 v trol. (d) Effect of COS and COS conjugates (100 μg/mL) on HIV-1 gp120 binding with host ceptor CD4 was investigated by ELISA. Binding was given as a relative percentage of the C group where gp120 and CD4 interaction did not interfere with any treatment. * p < 0.05, ** p and *** p < 0.001 vs. Control; # p < 0.05 COS-N vs. COS-Q.
The co-culture experiment was also performed to further understand the HIVinhibitory effect of COS-N and COS-Q. C8166 cells were co-cultured with HIV chronically infected H9 cells. The cell-to-cell interaction between the gp120 protein fected cells and CD4 receptors of infected cells were expected to mimic HIV-1 infec vivo and result in multinucleated giant cells and result in cell killing [30]. As seen in 4c, syncytia formation was inhibited by 58.24% after COS-N treatment (100 μg/m by 43.61% after COS-Q treatment (100 μg/mL). COS treatment was deemed ineffec protect cell-cell fusion. This result shows that COS-N and COS-Q treatment was inhibit cell-cell fusion. The entry stage of the HIV-1 viral cycle starts with the bind HIV-1 gp120 protein to a specific receptor on T cells called CD4 and its co-rec Therefore, the effect of COS conjugates on the cell-cell fusion was further invest Control. (d) Effect of COS and COS conjugates (100 µg/mL) on HIV-1 gp120 binding with host cell receptor CD4 was investigated by ELISA. Binding was given as a relative percentage of the Control group where gp120 and CD4 interaction did not interfere with any treatment. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. Control; # p < 0.05 COS-N vs. COS-Q.
The co-culture experiment was also performed to further understand the HIV-1 entry inhibitory effect of COS-N and COS-Q. C8166 cells were co-cultured with HIV-1IIIB chronically infected H9 cells. The cell-to-cell interaction between the gp120 protein of infected cells and CD4 receptors of infected cells were expected to mimic HIV-1 infection in vivo and result in multinucleated giant cells and result in cell killing [30]. As seen in Figure 4c, syncytia formation was inhibited by 58.24% after COS-N treatment (100 µg/mL) and by 43.61% after COS-Q treatment (100 µg/mL). COS treatment was deemed ineffective to protect cell-cell fusion. This result shows that COS-N and COS-Q treatment was able to inhibit cell-cell fusion. The entry stage of the HIV-1 viral cycle starts with the binding of HIV-1 gp120 protein to a specific receptor on T cells called CD4 and its co-receptors. Therefore, the effect of COS conjugates on the cell-cell fusion was further investigated with ELISA. As seen in Figure 4d, the COS-N and COS-Q treatment significantly hindered the binding of gp120 with CD4. DS was used as a positive control due to its reported HIV-1 entry inhibitory properties [27]. Although the inhibitory effect of COS conjugates was lower than that of DS (81.61%), the results indicate that the anti-HIV-1 effect of COS-N and COS-Q might exhibit itself by disrupting the interaction between the virus and host cell. Yoshida [38] suggests that the sulfate groups in sulfated polysaccharides might interact with positively charged amino acids in HIV-1 gp120. Similarly, Battulga et al. [4] reported the interaction between sulfated polysaccharides and HIV-1 surface protein, gp120. Although N-and Q-conjugated COS did not possess the negative charges of sulfation, Jeon and Kim [39] postulated that positive charges of COS amino acid conjugates might interact with negative charges on the cell surface. In this case, COS-N and COS-Q might compete with gp120 to bind the cell surface receptor-sulfated polysaccharides to inhibit viral entry as Crublet et al. [40] reported that gp120 of HIV-1 showed affinity to cell-associated sulfated groups, which consequently increased the viral infectivity. On the other hand, modification with polar amino acids is expected to increase the solubility of COS, and therefore, enhance the interaction between viral proteins and compounds. This was consistent with the present results which suggest that COS conjugated with amino acids inhibited HIV-1 infection by interfering with virus-host cell binding.

Materials
Chitosan oligomers (<1 kDa) were obtained from Kitto Life (Seoul, Korea). For the syntheses of COS derivatives N-conjugated with asparagine (N) and glutamine (Q), their N-t-tert-butyloxycarbonyl (Boc) amino acid derivatives were purchased from Sigma Chemical Co.

Addition of Amino Acids to COS
N-conjugation of asparagine and glutamine was carried out as previously reported [39]. Briefly, conjugates were prepared by the N-conjugation of Boc bound amino acids (Boc-AA) to the C-2 position of the chitosan oligomer (COS) monomers. Then, 25 g of COS with approx. 1 mM free amino group was dissolved in 50 mL water. Two hundred milliliters of methanol was added to COS and the pH of the mixture was adjusted to pH 6.8 with TEA. Mixture was put into shaker for 24 h after the addition of 100 mM Boc-AA and 100 mM dicyclohexyl carbodiimide (DCC) as the coupling agent. After 24 h, the mixture was filtered and kept overnight at 2 • C. Boc-AA-COS was removed from the mixture by precipitation via the addition of an adequate amount of ether, before being filtered and lyophilized. Finally, Boc protection was removed to obtain COS-AA. Asparagine-conjugated COS (COS-N) and glutamine-conjugated COS (COS-Q) were analyzed for the degree of substitution for COS-N: 0.78; and for COS-Q: 0.53, which were calculated from elemental analysis as described.

Cell Culture, HIV-1 Infection, Syncytia Formation, and Cell Viability Analysis
H9, H9/HIV-1 IIIB , and C8166 cell lines were propagated at 37°C under 5% CO 2 in complete RPMI 1640 medium supplemented with 10% FBS, 100 µg of streptomycin per ml, and 100 U of penicillin per ml. All cells were cultured in either T25 or T75 cell culture flasks. Cells were sub-cultured 2-3 times a week (1 × 10 5 cells/T-25 flask). Cells were discarded every 2 months and replaced from fresh stocks. Prior to experiments, cells were transferred into 48-well plates unless otherwise noted.
HIV-1 infection of human T cell line C8166 cell was followed by large, multinucleated giant cell formations called syncytia. These syncytia formations eventually led to cell death after swelling. Basically, C8166 cells were seeded in 48-well plates (1 × 10 5 cells/well) in the presence or absence of different concentrations of COS conjugates in RPMI-1640 with 5% FBS. Cells were infected after 2 h incubation with HIV-1 RF (100 µL) diluted in RPMI-1640 to 200 CCID 50 (µg/mL). Cells were kept at 37 • C for 48 h and syncytia was confirmed and counted optically using an inverted microscope (DMire2, Leica Microsystems, Solms, Germany). For each treatment group, three wells were counted thrice and the average ± SD was noted as the final syncytia count.
To determine the protective effect of amino acid-conjugated COS against HIV-1 induced lysis in C8166 cells, an MTT-formazan-based cell viability assay was performed. Cells in log-growth phase (four days post-seeding) were harvested, washed, and added in 48-well plate (1 × 10 5 cells/well) in the presence or absence of different concentrations of COS conjugates. Stock supernatants of HIV-1 IIIB were diluted RPMI-1640 to yield sufficient cytopathicity (~90% cell kill in 5 days) (CCID 50 ) and used to infect the cells (100 µL per well). Plates were kept for 5 days at 37 • C and 100 µL MTT solution (500 µg/mL in PBS) was added to each well and cells were kept for a further 4 h at 37 • C. The formazan salt formed by viable cells was dissolved in acidified propanol containing 50% DMSO and 4% triton X-100. The absorbance values of each well were measured at 540 nm with a GENios microplate reader (Tecan, Austria). The absorbance value of untreated and uninfected cells was taken as 100% comparison viability and rest of the treatment groups were given as relative percentage.

Measurement of p24 Antigen
To measure the p24 antigen levels in HIV-1-infected cell culture medium, a commercial p24 antigen capture ELISA was used (Perkin-Elmer Life Sciences, Boston, MA, USA) following the manufacturer's directions. Basically, H9 cells were cultured in 24-well plates at a density of 5 × 10 5 cells/well and infected with HIV-1 RF as described in Section 3.3. Cells were treated with COS-conjugates for five days and the supernatants of the culture wells were used to detect the virus released to medium by p24 ELISA.
Expression of p24 protein was investigated by Western blotting in cellular fractions and culture medium. H9 cells were cultured and infected as described above. Cells were treated with COS-conjugates for five days. To analyze the protein levels of p24, the total protein was isolated from cells through the addition of 1 mL lysis buffer containing 50 mM Tris-HCl, 0.4% (w/v) NP-40, 120 mM NaCl, 1.5 mM MgCl 2 , 2 mM PMSF, 3 mM NaF, and 1 mM DTT to each well after harvesting the supernatant. Viral proteins were obtained from harvested supernatants as described earlier [27]. On the other hand, cell lysates were centrifuged at 12,000 rpm for 10 min and the supernatants were used for Western blotting. The protein concentration of the samples was calculated using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and twenty micrograms of protein from each well was loaded on a 10% SDS-PAGE gel. Following SDS-PAGE, the proteins were transferred to nitrocellulose membranes. Blotted membranes were then blocked in 5% skim milk for 4 h at room temperature and hybridized with p24 monoclonal primary antibody overnight at 4 • C. Membranes were then subjected to horse radish peroxidase conjugated anti-mouse antibody for 1 h at room temperature. Protein bands on membranes were visualized with a commercial chemiluminescence kit (Amersham ECL detection kit, GE Healthcare, Chicago, IL, USA) and the images were taken on a Fujifilm Imaging System (Fujifilm Life Science, Tokyo, Japan).

HIV-1 RT and Protease Activity Assay
The activity of HIV-1 reverse transcriptase, isolated from the virus pellet of H9/HIV-1 IIIB culture supernatant as described in earlier studies [27], was evaluated using a fluores-cence RT assay kit (EnzChek, InvitroGen, Carslbad, CA, USA) according to the manufacturer's protocol. The activity of the HIV-RT was measured as the fluorescence intensity of the wells as a result of RT activity on the substrate. Fluorescence intensity was measured at 480 nm (excitation) and 520 nm (emission) with a GENios microplate reader (Tecan Austria GmbH, Grodig, Austria) after the addition of 173 µL of fluorescent PicoGreen reagent prepared in TE buffer.
In order to assess the protease inhibitory effect of COS conjugates, a commercial SensoLyte 520 HIV-1 protease assay kit (Anaspec, CA, USA) was used according to manufacturer's directions. COS conjugates were tested for their ability to inhibit the proteolytic cleavage of HiLyte Fluor™488/QXL™520 FRET peptide (obtained from the SensoLyte 520 HIV-1 protease assay kit) by HIV-1 protease. The amount of HiLyte Fluor™488 produced by successful protease activity was measured by GENios ® microplate reader (Tecan Austria GmbH, Austria).

Delayed Addition and Co-Culture Assays
C8166 cells were seeded in a 48-well plate (3 × 10 4 cells/well). Cells were infected as previously described in Section 3.3 (syncytia formation count). After the addition of the virus, 100 µL of 100 µg/mL COS conjugates, saquinavir, and azidothymidine were added into multiple wells. Blank group was only treated with samples and incubated without being infected with the virus. After a total of 96 h incubation, cellular viability was assessed using the MTT assay, as previously described in Section 3.3, and the protection ability is calculated in reference to treated yet uninfected cell groups.
For the co-culture assays, C8166 and H9 cells were cultured together. Briefly, C8166 cells were seeded in a 48-well plate (5 × 10 4 cell/well) along with H9 cells (infected with HIV-1 IIIB as described in Section 3.3) in the ratio of 10:1 in the presence or absence of COS conjugates. The cells were incubated for 48 h, and the number of syncytia was counted using a light microscope as noted in Section 3.3.

Analysis of gp120-CD4 Binding
The effect of COS conjugates on HIV-1 entry was investigated via its interaction with CD4-gp120 binding. ELISA was carried out as previously reported with the required modifications [30]. Briefly, the wells of an ELISA plate were coated with 2 µg/mL anti-gp120 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in carbonate buffer (pH 9.6) and incubated overnight at 4 • C. Wells were then blocked with PBS containing 0.1% BSA at 37 • C for 1 h. Recombinant HIV-1 IIIB gp120 protein (100 ng/well) in PBS was added to each well and the plate was incubated at 37 • C for 1 h. The wells were then washed with PBS containing 0.05% Tween 20. Different concentrations of PCOS were added to each well along with human sCD4 (100 ng/well) and the plates were further incubated at 37 • C for 1 h. After the incubation wells were treated with anti-sCD4 IgG (250 ng/mL), another incubation followed at 37 • C for 1 h. Subsequently, the horseradish peroxidase conjugated with streptavidin was added for the detection of gp120-bound CD4 proteins. Measurement was carried out by detecting the absorbance values of wells at 405 nm after adding a water-soluble horseradish peroxidase substrate that produces the yellow color (O-phenylenediamine dihydrochloride).

Statistical Analysis
The statistical significance of experimental data was determined and expressed as a mean of three independent experiments ± standard deviation (SD). Differences between the means were analyzed using the analysis of variance (ANOVA) procedure of Statistical Analysis System, SAS v9.2 (SAS Institute, Cary, NC, USA) with Duncan's multiple range test as a post hoc analysis. The significance of differences was defined at the p < 0.05 level.

Conclusions
In conclusion, the current results point out that COS-N and COS-Q possibly inhibited HIV-1 infection by blocking the connection between HIV-1 (or infected cells) and uninfected cells. However, DS and the other positive controls showed higher activity than COS conjugates, but the efficiency of COS conjugates did not generate further interest in developing them into antiviral agents. Nevertheless, the experiments showed that the N-conjugation of COS with amino acids enhances its antiviral capabilities and the amide group side chains added COS anti-HIV-1 properties. Further studies with longer peptide sequence conjugations and experiments that aim to elucidate the interaction between COS conjugates and the HIV-1 cell entry mechanism should yield valuable data towards the utilization of COS-protein conjugates as lead molecules for HIV-1 drug development. Data Availability Statement: Data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The author declares no conflict of interest.