Safety and immunogenicity of a polyvalent DNA-protein HIV vaccine with matched Env immunogens delivered as a prime-boost regimen or coadministered in HIV-uninfected adults in the USA (HVTN 124): a phase 1, placebo-controlled, double-blind randomised controlled trial.

BACKGROUND: An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. METHODS: HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. FINDINGS: Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. INTERPRETATION: Both vaccine regimens were safe, warranting evaluation in larger trials. FUNDING: US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.

VXX-401, a novel anti-PCSK9 vaccine, reduces LDL-C in cynomolgus monkeys.

Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of disease burden in the world and is highly correlated with chronic elevations of LDL-C. LDL-C-lowering drugs, such as statins or monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9), are known to reduce the risk of cardiovascular diseases; however, statins are associated with limited efficacy and poor adherence to treatment, whereas PCSK9 inhibitors are only prescribed to a "high-risk" patient population or those who have failed other therapies. Based on the proven efficacy and safety profile of existing monoclonal antibodies, we have developed a peptide-based vaccine against PCSK9, VXX-401, as an alternative option to treat hypercholesterolemia and prevent ASCVD. VXX-401 is designed to trigger a safe humoral immune response against PCSK9, resulting in the production of endogenous antibodies and a subsequent 30-40% reduction in blood LDL-C. In this article, VXX-401 demonstrates robust immunogenicity and sustained serum LDL-C-lowering effects in nonhuman primates. In addition, antibodies induced by VXX-401 bind to human PCSK9 with high affinity and block the inhibitory effect of PCSK9 on LDL-C uptake in a hepatic cell model. A repeat-dose toxicity study conducted in nonhuman primates under good laboratory practices toxicity indicated a suitable safety and tolerability profile, with injection site reactions being the main findings. As a promising safe and effective LDL-C-lowering therapy, VXX-401 may represent a broadly accessible and convenient option to treat hypercholesterolemia and prevent ASCVD.

Human CD4-Binding Site Antibody Elicited by Polyvalent DNA Prime-Protein Boost Vaccine Neutralizes Cross-Clade Tier-2-HIV Strains.

The vaccine elicitation of HIV-neutralizing antibodies with tier-2-neutralization breadth has been a challenge. Here, we report the isolation and characteristics of a CD4-binding site specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent gp120 DNA prime-protein boost vaccine. HmAb64 derived from heavy chain variable germline gene IGHV1-18, light chain germline gene IGKV1-39, and had a 3rd heavy chain complementarity determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 21 (10%), including tier-2 neutralization resistant strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 bound to a conformation between prefusion closed and occluded open forms of envelope trimer, using both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4-binding site. A gp120 subunit-based vaccine can thus elicit an antibody capable of tier 2-HIV neutralization.

RBD-Protein/Peptide Vaccine UB-612 Elicits Mucosal and Fc-Mediated Antibody Responses against SARS-CoV-2 in Cynomolgus Macaques.

Antibodies provide critical protective immunity against COVID-19, and the Fc-mediated effector functions and mucosal antibodies also contribute to the protection. To expand the characterization of humoral immunity stimulated by subunit protein-peptide COVID-19 vaccine UB-612, preclinical studies in non-human primates were undertaken to investigate mucosal secretion and the effector functionality of vaccine-induced antibodies in antibody-dependent monocyte phagocytosis (ADMP) and antibody-dependent NK cell activation (ADNKA) assays. In cynomolgus macaques, UB-612 induced potent serum-neutralizing, RBD-specific IgG binding, ACE2 binding-inhibition antibodies, and antibodies with Fc-mediated effector functions in ADMP and ADNKA assays. Additionally, immunized animals developed mucosal antibodies in bronchoalveolar lavage fluids (BAL). The level of mucosal or serum ADMP and ADNKA antibodies was found to be UB-612 dose-dependent. Our results highlight that the novel subunit UB-612 vaccine is a potent B-cell immunogen inducing polyfunctional antibody responses contributing to anti-viral immunity and vaccine efficacy.

A novel RBD-protein/peptide vaccine elicits broadly neutralizing antibodies and protects mice and macaques against SARS-CoV-2.

The development of safe and effective vaccines to respond to COVID-19 pandemic/endemic remains a priority. We developed a novel subunit protein-peptide COVID-19 vaccine candidate (UB-612) composed of: (i) receptor binding domain of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc; (ii) five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins (three from S2 subunit, one from membrane and one from nucleocapsid), and one universal Th peptide; (iii) aluminum phosphate as adjuvant. The immunogenicity and protective immunity induced by UB-612 vaccine were evaluated in four animal models: Sprague-Dawley rats, AAV-hACE2 transduced BALB/c mice, rhesus and cynomolgus macaques. UB-612 vaccine induced high levels of neutralizing antibody and T-cell responses, in all animals. The immune sera from vaccinated animals neutralized the SARS-CoV-2 original wild-type strains and multiple variants of concern, including Delta and Omicron. The vaccination significantly reduced viral loads, lung pathology scores, and disease progression after intranasal and intratracheal challenge with SARS-CoV-2 in mice, rhesus and cynomolgus macaques. UB-612 has been tested in primary regimens in Phase 1 and Phase 2 clinical studies and is currently being evaluated in a global pivotal Phase 3 clinical study as a single dose heterologous booster.

High Neutralizing Antibody Levels Against Severe Acute Respiratory Syndrome Coronavirus 2 Omicron BA.1 and BA.2 After UB-612 Vaccine Booster.

The highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has caused high rates of breakthrough infections in those previously vaccinated with ancestral strain coronavirus disease 2019 (COVID-19) vaccines. Here, we demonstrate that a booster dose of UB-612 vaccine candidate delivered 7-9 months after primary vaccination increased neutralizing antibody levels by 131-, 61-, and 49-fold against ancestral SARS-CoV-2 and the Omicron BA.1 and BA.2 variants, respectively. Based on the receptor-binding domain protein binding antibody responses, the UB-612 third-dose booster may lead to an estimated approximately 95% efficacy against symptomatic COVID-19 caused by the ancestral strain. Our results support UB-612 as a potential potent booster against current and emerging SARS-CoV-2 variants.

Broadly binding and functional antibodies and persisting memory B cells elicited by HIV vaccine PDPHV.

Since publishing our original reports on the safety and immunogenicity of a polyvalent DNA prime-protein boost HIV vaccine (PDPHV) which elicited high titer antibody responses with broad specificity, neutralizing activities to multiple HIV-1 subtypes, as well as poly-functional T cell responses, accumulated findings from other HIV vaccine studies indicated the important roles of Ig isotype distribution, Fc medicated functions and the persistence of memory immune responses which were not studied in previous PDPHV related reports. The current report provides further detailed characterization of these parameters in human volunteers receiving the PDPHV regimen. Antibody responses were assessed using IgG isotype and gp70-V1V2-binding ELISAs, peptide arrays, and antibody-dependent cellular cytotoxicity (ADCC) assays. B cell ELISPOT was used to detect gp120-specific memory B cells. Our results showed that the gp120-specific antibodies were primarily of the IgG1 isotype. HIV-1 envelope protein variable regions V1 and V2 were actively targeted by the antibodies as determined by specific binding to both peptide and V1V2-carrying scaffolds. The antibodies showed potent and broad ADCC responses. Finally, the B cell ELISPOT analysis demonstrated persistence of gp120-specific memory B cells for at least 6 months after the last dose. These data indicate that broadly reactive binding Abs and ADCC responses as well as durable gp120-specific memory B cells were elicited by the polyvalent heterologous prime-boost vaccination regimens and showed great promise as a candidate HIV vaccine.

Non-neutralizing antibodies targeting the immunogenic regions of HIV-1 envelope reduce mucosal infection and virus burden in humanized mice.

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity.

DNA priming immunization is more effective than recombinant protein vaccine in eliciting antigen-specific B cell responses.

While DNA prime-protein boost vaccination approach has been widely used in preclinical and clinical studies especially in the field of HIV vaccine development, the exact role of DNA immunization has not been fully identified. Our previous work demonstrated that DNA immunization was able to elicit T follicular helper (Tfh) cell responses and germinal center (GC) B cell development in a mouse model. In the current report, a mouse immunogenicity study was conducted to further ask whether DNA immunization is able to elicit antigen-specific B cell responses. Using HIV-1 Env as model antigen delivered in the form of DNA prime-protein boost, our data demonstrated that DNA prime was able to enhance the antigen-specific B cell responses for both Env-specific antibody secreting cells (ASC) and memory B cells. Furthermore, the DNA priming can greatly reduce the need of including an adjuvant as part of the recombinant protein vaccine boost formulation. Our findings revealed one mechanism that supports the value of DNA priming in assisting the inductin of high affinity and long lasting antigen specific antibody responses.

A novel DNA and protein combination COVID-19 vaccine formulation provides full protection against SARS-CoV-2 in rhesus macaques.

The current study aims to develop a safe and highly immunogenic COVID-19 vaccine. The novel combination of a DNA vaccine encoding the full-length Spike (S) protein of SARS-CoV-2 and a recombinant S1 protein vaccine induced high level neutralizing antibody and T cell immune responses in both small and large animal models. More significantly, the co-delivery of DNA and protein components at the same time elicited full protection against intratracheal challenge of SARS-CoV-2 viruses in immunized rhesus macaques. As both DNA and protein vaccines have been proven safe in previous human studies, and DNA vaccines are capable of eliciting germinal center B cell development, which is critical for high-affinity memory B cell responses, the DNA and protein co-delivery vaccine approach has great potential to serve as a safe and effective approach to develop COVID-19 vaccines that provide long-term protection.

Identification of key genes involved in tamoxifen-resistant breast cancer using bioinformatics analysis.

BACKGROUND: The purpose of the present study was to investigate the molecular mechanisms of tamoxifen resistance in breast cancer and to identify potential targets for antitamoxifen resistance. METHODS: Differentially expressed genes (DEGs) in tamoxifen-resistant and tamoxifen-sensitive breast cancer cells were assessed using the GSE67916 dataset acquired from the Gene Expression Omnibus database. Gene ontology (GO) and pathway enrichment analyses were applied to investigate the functions and pathways of the DEGs. Subsequently, the protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING), and subnetworks were further analyzed by Molecular Complex Detection (MCODE). The PPI network and subnetworks were visualized using Cytoscape software. RESULTS: In total, 438 DEGs were identified, of which 300 were upregulated and 138 were downregulated. The DEGs were significantly enriched in the protein binding, cellular response to estradiol stimulus, and immune response GO terms while the most significant pathways included the mitogen-activated protein kinase (MAPK) signaling pathway in cancer. The PPI network of DEGs was constructed with 288 nodes and 629 edges, and 2 subnetworks were screened out from the entire network. CONCLUSIONS: A number of significant hub DEGs were identified based on their degree of connectivity in the PPI network, , included MAPK1 (node degree 36), ESR1 (node degree 27), SMARCA4 (node degree 27), RANBP2 (node degree 25), and PRKCA (node degree 21). These critical hub genes were found to be related to tamoxifen resistance in breast cancer. The results of this study further the understanding of tamoxifen resistance at the molecular level and identify potential therapeutic targets for tamoxifen-resistant breast cancer.

The values and limitations of mathematical modelling to COVID-19 in the world: a follow up report.

We previously described a mathematical model to simulate the course of the COVID-19 pandemic and try to predict how this outbreak might evolve in the following two months when the pandemic cases will drop significantly. Our original paper prepared in March 2020 analyzed the outbreaks of COVID-19 in the US and its selected states to identify the rise, peak, and decrease of cases within a given geographic population, as well as a rough calculation of accumulated total cases in this population from the beginning to the end of June 2020. The current report will describe how well the later actual trend from March to June fit our model and prediction. Similar analyses are also conducted to include countries other than the US. From such a wide global data analysis, our results demonstrated that different US states and countries showed dramatically different patterns of pandemic trend. The values and limitations of our modelling are discussed.

A method for isotope exchange kinetics in mineral-water system-an in-situ study on oxygen isotope exchange in Na2CO3-H2O system.

The experiments on the exchange reaction rate of the oxygen isotopes in the mineral-water system with initial conditions of 100 °C and 240 MPa, 100 °C and 924 MPa, 118 °C and 170 MPa are taken. The oxygen isotope 18O exchange reactions between aqueous C16O32- and H218O are mainly traced by measuring the Raman peak intensity of oxygen-containing elements (CO32-) in sodium carbonate solution. The inconsistent trends between the molar fraction and the concentration of C18O216O2- indicate oxygen isotope exchange between sodium carbonate and heavy water accompanied by dissolution and recrystallization between supersaturated sodium carbonate solution and solid sodium carbonate. In the heterogeneous experimental systems the order of oxygen isotope exchange reactions are more than 1 and the dynamics of oxygen isotope exchange conform to JMAK kinetics model with the nucleation and growth processes of sodium carbonate crystal.

Mathematic modeling of COVID-19 in the United States.

COVID-19, the worst pandemic in 100 years, has rapidly spread to the entire world in 2 months since its early report in January 2020. Based on the publicly available data sources, we developed a simple mathematic modeling approach to track the outbreaks of COVID-19 in the US and three selected states: New York, Michigan and California. The same approach is applicable to other regions or countries. We hope our work can stimulate more effort in understanding how an outbreak is developing and how big a scope it can be and in what kind of time framework. Such information is critical for outbreak control, resource utilization and re-opening of the normal daily life to citizens in the affected community.

Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions.

Envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here, we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE), produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under good manufacturing practice (GMP) conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between the two GMP lots. Our data confirmed previous studies in the field, showing an abundance of oligomannose on Env protein, with 40 to 50% of glycans being Man5 to Man9 on all four proteins under all production conditions. Overall, the differences in occupancy and glycan forms among different Env subtypes produced under different conditions were less dramatic than anticipated, and antigenicity analysis with a panel of six monoclonal antibodies, including antibodies that recognize glycan forms, showed that all four gp120s maintained their antibody-binding profiles. Such findings have major implications for the final production of a clinical HIV vaccine with Env glycoprotein components.IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.

Author Correction: The wide utility of rabbits as models of human diseases.

This article was originally published under a CC BY-NC-SA License, but has now been made available under a CC BY 4.0 License.

Preparation of porcine enterotoxigenic Escherichia coli (ETEC) ghosts and immunogenic analysis in a mouse model.

Enterotoxignenic Escherichia coli (ETEC)-associated colibacillosis causes high levels of morbidity and mortality in neonatal piglets. Vaccination is among effective strategy to fight against ETEC-related diseases. Bacterial ghosts (BGs) are empty bacterial envelopes, which substain subtle antigenic comformation in bacterial outer membrane. In this study, a BG vaccine was generated using porcine ETEC isolated strain DQ061 and evaluated its safety and immunogenicity in a mouse model. The recombinant bacteria were constructed by transformation of lysis plasmid pHH43 and generation of BGs was conducted in a lysis rate of 99.93% by incubation of the recombinant bacteria at 42 °C for 2 h. Mice were immunized subcutaneously twice in 2-week intervals with BGs, BGs emulsified with ISA 206 adjuvant, or formalin-inactivated ETEC vaccine after safety test. Mice with either of two BG vaccines developed higher titer of antibodies, secreted higher titer of interleukin 4, gamma interferon and alpha tumor necrosis factor after 2 doses than those with formalin-inactivated ETEC vaccine or those with adjuvant placebo (P < 0.01). The quantity of CD4+ and CD8+ T lymphocyte in spleen was higher in both BG groups than that in the inactivated vaccine group or adjuvant group 2 weeks post boost immunization (P < 0.05). The vaccinated mice were challenged intraperitoneally with 10 × LD50 dose of DQ061. Mice with the BGs plus adjuvant were completely protected against challenge, compared to 60% protection of mice with the inactivated vaccine. Mice exhibited decreased tissue lesion and reduced bacterial loads in the BGs groups by comparison with those with the inactivated vaccine or adjuvant only. Our results validated that the ETEC BGs bear high safety and immunogenicity in a mouse model, suggesting a potential of further evaluation in a pig model.

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4ß7.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

Using DNA Immunization to Elicit Monoclonal Antibodies in Mice, Rabbits, and Humans.

In recent vaccine studies, DNA immunization was found to effectively stimulate both innate and adaptive immunities to elicit high levels of antigen-specific antibody responses. The DNA molecule itself can activate multiple pathways of innate immunity. The in vivo production of antigens allows for presentation by major histocompatibility complexes, so that T-cell responses are generated to help in the development of antigen-specific B cells, leading to high-affinity antibody response. By using the same process, DNA immunization should also be effective at producing functionally potent monoclonal antibodies (mAbs). Furthermore, the in vivo expressed proteins can maximally maintain the native structures and go through appropriate post-transcriptional modifications. By combining such advantages, DNA immunization can be expected to play more important roles in the future to elicit mAbs against difficult targets from a wide range of host systems. The current report shares our group's experience in using DNA immunization to elicit mAbs in the mouse, rabbit, and human models.

Structural Comparison of Human Anti-HIV-1 gp120 V3 Monoclonal Antibodies of the Same Gene Usage Induced by Vaccination and Chronic Infection.

Elucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (MAbs) encoded by the same immunoglobulin genes at different stages of maturation can help to elucidate the maturation process. We have analyzed four human anti-V3 MAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two MAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime/protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients. The somatic hypermutation (SHM) rates in VH of vaccine-induced MAbs are lower than in chronic HIV infection-induced MAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these MAbs bind the V3 epitope with a new cradle-binding mode and that the V3 ß-hairpin lies along the antigen-binding groove, which consists of residues from both heavy and light chains. Residues conserved from the germ line sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity. Our data provide a unique example of germ line sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection.IMPORTANCE Understanding the structural basis of gene usage and affinity maturation for anti-HIV-1 antibodies may help vaccine design and development. Antibodies targeting the highly immunogenic third variable loop (V3) of HIV-1 gp120 provide a unique opportunity for detailed structural investigations. By comparing the sequences and structures of four anti-V3 MAbs at different stages of affinity maturation but of the same V gene usage, two induced by vaccination and another two by chronic infection, we provide a fine example of how germ line sequence determines the essential elements for epitope recognition and how affinity maturation improves the antibody's recognition of its epitope.