A Glycoconjugate Antigen Based on the Recognition Motif of a Broadly Neutralizing Human Immunodeficiency Virus Antibody, 2G12, Is Immunogenic but Elicits Antibodies Unable To Bind to the Self Glycans of gp120▿

The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man4) that corresponds to the D1 arm of Man9GlcNAc2 inhibits 2G12 binding to gp120 as efficiently as Man9GlcNAc2 itself, indicating the potential use of Man4 as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man4 on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man4 up to a limit which is achieved at ∼10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man4)14 elicits significant serum Ab titers to Man4. However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man9 derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man4 on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of cross-reacting with gp120 and HIV-1

Defining criteria for oligomannose immunogens for HIV using icosahedral virus capsid scaffolds

SummaryThe broadly neutralizing antibody 2G12 recognizes a conserved cluster of high-mannose glycans on the surface envelope spike of HIV, suggesting that the “glycan shield” defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semisynthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Qβ and cowpea mosaic virus (CPMV). The Qβ glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, antibodies against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titers of anti-mannose antibodies that do not crossreact with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV

Cryopreservation of human mucosal tissues.

BackgroundCryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.Methods and findingsTo find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.ConclusionsCryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar

Cryopreservation of human mucosal tissues

<div><p>Background</p><p>Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol.</p><p>Methods and findings</p><p>To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated “cryopreservation”) and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol (“vitrification”). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59–84%]) than before (50% [38–62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones.</p><p>Conclusions</p><p>Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.</p></div

Cryopreserved colorectal explants are somewhat less susceptible to HIV infection than fresh colorectal explants, while cryopreserved and fresh ectocervical explants are similarly susceptible.

<p><b>A</b>, HIV challenge curves for colorectal tissue showing HIV p24 concentrations (picograms/milliliter) from a single tissue donor either fresh or after cryopreservation with the indicated cryopreservation media. Each line represents a single explant and points represent supernatant collections. All three explants come from the same donor. “DMSO”, dimethylsulfoxide; “EG”, “ethylene glycol”; “mM”, “millimolar”. <b>B</b>, Summary of HIV challenge experiments for colorectal tissues. Each square represents a single explant, with each column representing a different donor (total n = 12 donors with 2–3 explants per donor per condition, as shown). Light squares indicate explants that became infected and dark squares explants that did not. Missing squares indicate cases where there was inadequate tissue available to have three explants in all conditions or where an explant was lost by experimental error. Explants are defined as infected on the first day after day 5 where the p24 concentration exceeds 250 pg/mL. <b>C</b>, Proportion of colorectal explants that became infected. Points show the proportion of all challenged explants that became infected. Lines show the 95% binomial proportion confidence intervals. <b>D</b>, Survival curves indicating cumulative HIV infection for all colorectal explants tested in each condition. Fresh explants are shown in green, cryopreserved with 10% DMSO in orange, and cryopreserved with 6% DMSO, 5% EG, and 50 mM trehalose in blue. <b>E</b>, HIV challenge curves for ectocervical tissue, as in A. <b>F</b>, Summary of HIV challenge experiments for ectocervical tissue, as in B, with a threshold of infection of 500 pg/mL (total n = 11 donors with 2–3 explants per donor per condition, as shown). <b>G</b>, Proportion of ectocervical explants that became infected, as in C. <b>H</b>, Survival curves for HIV infection of ectocervical explants, as in D.</p

Ten-fold more cells are recovered after cryopreservation than vitrification of colorectal biopsies.

<p><b>A</b>, Number of live cells recovered after digestion of colorectal biopsies that had been cryopreserved or vitrified (n = 5 donors with 15 biopsies per donor per condition). <b>B</b>, Cell phenotypes as measured by flow cytometry. Leukocytes are defined as CD45⁺. T cells are CD45⁺CD3⁺. Monocytes are CD45⁺CD3^-CD13⁺. CD33 myeloid are CD45⁺CD3^-CD33⁺. Granulocytes are CD45⁺CD3^-CD66b⁺. Small gray symbols indicate the number (A) or percent (B) from fifteen colorectal biopsies, with gray lines indicating samples from the same tissue donor. Black symbols show the mean across all tissue donors and black vertical lines show the 95% confidence interval of the mean.</p

Retention of antiretroviral drugs in ectocervical and colorectal tissue after cryopreservation.

<p><b>A</b>, Concentration of drug measured in tissues either fresh or after cryopreservation (n = 3 tissue donors per condition with two explants per donor per condition). Explants were exposed in vitro to antiretroviral drugs at the concentrations indicated above each plot. After drug exposure and cryopreservation, drug levels were measured in the tissues. Small gray symbols indicate the average of two explants and gray lines connect samples from the same tissue donor (dashed indicates colorectal, solid indicates ectocervical). Large symbols indicate the mean for that tissue type (orange indicates colorectal, green indicates ectocervical). Vertical green and orange lines show 95% confidence intervals. <b>B</b>, Normalized drug concentration expressed as percent of drug concentration in fresh samples from the same tissue donor. Colored symbols show the mean across all tissue donors and colored vertical lines show the 95% confidence interval of the mean. The mean recovery for dapivirine in colorectal tissue is off-scale and the value is indicated in text.</p