Metabolomic Signatures Differentiate Immune Responses in Avian Influenza Vaccine Recipients.

BACKGROUND: Avian influenza viruses pose significant risk to human health. Vaccines targeting the hemagglutinin of these viruses are poorly immunogenic without the use of adjuvants. METHODS: Twenty healthy men and women (18-49 years of age) were randomized to receive two doses of inactivated influenza A/H5N1 vaccine alone (IIV) or with AS03 adjuvant (IIV-AS03) one month apart. Urine and serum samples were collected on day 0 and on days 1, 3, and 7 following first vaccination and subjected to metabolomics analyses to identify metabolites, metabolic pathways, and metabolite clusters associated with immunization. RESULTS: Seventy-three differentially abundant (DA) serum and 88 urine metabolites were identified for any post-vaccination day comparison. Pathway analysis revealed enrichment of tryptophan, tyrosine and nicotinate metabolism in urine and serum among IIV-AS03 recipients. Increased urine abundance of 4-vinylphenol sulfate on Day 1 was associated with serologic response based on hemagglutination inhibition responses. In addition, 9 DA urine metabolites were identified in participants with malaise compared to those without. CONCLUSIONS: Our findings suggest that tryptophan, tyrosine, and nicotinate metabolism are upregulated among IIV-AS03 recipients compared with IIV alone. Metabolites within these pathways may serve as measures of immunogenicity and may provide mechanistic insights for adjuvanted vaccines.

Distinct gene expression signatures comparing latent tuberculosis infection with different routes of Bacillus Calmette-Guérin vaccination.

Tuberculosis remains an international health threat partly because of limited protection from pulmonary tuberculosis provided by standard intradermal vaccination with Bacillus of Calmette and Guérin (BCG); this may reflect the inability of intradermal vaccination to optimally induce pulmonary immunity. In contrast, respiratory Mycobacterium tuberculosis infection usually results in the immune-mediated bacillary containment of latent tuberculosis infection (LTBI). Here we present RNA-Seq-based assessments of systemic and pulmonary immune cells from LTBI participants and recipients of intradermal and oral BCG. LTBI individuals uniquely display ongoing immune activation and robust CD4 T cell recall responses in blood and lung. Intradermal BCG is associated with robust systemic immunity but only limited pulmonary immunity. Conversely, oral BCG induces limited systemic immunity but distinct pulmonary responses including enhanced inflammasome activation potentially associated with mucosal-associated invariant T cells. Further, IL-9 is identified as a component of systemic immunity in LTBI and intradermal BCG, and pulmonary immunity following oral BCG.

Phase 1 Open-Label Dose Escalation Trial for the Development of a Human Bacillus Calmette-Guérin Challenge Model for Assessment of Tuberculosis Immunity In Vivo.

BACKGROUND: A controlled human infection model for assessing tuberculosis (TB) immunity can accelerate new vaccine development. METHODS: In this phase 1 dose escalation trial, 92 healthy adults received a single intradermal injection of 2 × 106 to 16 × 106 colony-forming units of Bacillus Calmette-Guérin (BCG). The primary endpoints were safety and BCG shedding as measured by quantitative polymerase chain reaction, colony-forming unit plating, and MGIT BACTEC culture. RESULTS: Doses up to 8 × 106 were safe, and there was evidence for increased BCG shedding with dose escalation. The MGIT time-to-positivity assay was the most consistent and precise measure of shedding. Power analyses indicated that 10% differences in MGIT time to positivity (area under the curve) could be detected in small cohorts (n = 30). Potential biomarkers of mycobacterial immunity were identified that correlated with shedding. Transcriptomic analysis uncovered dose- and time-dependent effects of BCG challenge and identified a putative transcriptional TB protective signature. Furthermore, we identified immunologic and transcriptomal differences that could represent an immune component underlying the observed higher rate of TB disease incidence in males. CONCLUSIONS: The safety, reactogenicity, and immunogenicity profiles indicate that this BCG human challenge model is feasible for assessing in vivo TB immunity and could facilitate the vaccine development process. CLINICAL TRIALS REGISTRATION: NCT01868464 (ClinicalTrials.gov).

Corrigendum: The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

[This corrects the article DOI: 10.3389/fimmu.2022.1093242.].

Utility of targeted gene sequencing to differentiate myeloid malignancies from other cytopenic conditions.

The National Heart, Lung, and Blood Institute-funded National MDS Natural History Study (NCT02775383) is a prospective cohort study enrolling patients with cytopenia with suspected myelodysplastic syndromes (MDS) to evaluate factors associated with disease. Here, we sequenced 53 genes in bone marrow samples harvested from 1298 patients diagnosed with myeloid malignancy, including MDS and non-MDS myeloid malignancy or alternative marrow conditions with cytopenia based on concordance between independent histopathologic reviews (local, centralized, and tertiary to adjudicate disagreements when needed). We developed a novel 2-stage diagnostic classifier based on mutational profiles in 18 of 53 sequenced genes that were sufficient to best predict a diagnosis of myeloid malignancy and among those with a predicted myeloid malignancy, predict whether they had MDS. The classifier achieved a positive predictive value (PPV) of 0.84 and negative predictive value (NPV) of 0.8 with an area under the receiver operating characteristic curve (AUROC) of 0.85 when classifying patients as having myeloid vs no myeloid malignancy based on variant allele frequencies (VAFs) in 17 genes and a PPV of 0.71 and NPV of 0.64 with an AUROC of 0.73 when classifying patients as having MDS vs non-MDS malignancy based on VAFs in 10 genes. We next assessed how this approach could complement histopathology to improve diagnostic accuracy. For 99 of 139 (71%) patients (PPV of 0.83 and NPV of 0.65) with local and centralized histopathologic disagreement in myeloid vs no myeloid malignancy, the classifier-predicted diagnosis agreed with the tertiary pathology review (considered the internal gold standard).

The antibody landscapes following AS03 and MF59 adjuvanted H5N1 vaccination.

Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays (NCT01317758 and NCT01317745). The arrays were designed to reflect both full-length and globular head HA derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and heterosubtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. The adjuvanted vaccine was also associated with the elicitation of stalk-directed antibody. We established good correlation of the array antibody responses to H5 antigens with standard HA inhibition and microneutralization titers.

Failure to Detect Mutations in U2AF1 due to Changes in the GRCh38 Reference Sequence.

The U2AF1 gene is a core part of mRNA splicing machinery and frequently contains somatic mutations that contribute to oncogenesis in myelodysplastic syndrome, acute myeloid leukemia, and other cancers. A change introduced in the GRCh38 version of the human reference build prevents detection of mutations in this gene, and others, by variant calling pipelines. This study describes the problem in detail and shows that a modified GRCh38 reference build with unchanged coordinates can be used to ameliorate the issue.

The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

Introduction: Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial. Methods: We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths. Results and Discussion: Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

Human Antibody Responses Following Vaccinia Immunization Using Protein Microarrays and Correlation With Cell-Mediated Immunity and Antibody-Dependent Cellular Cytotoxicity Responses.

BACKGROUND: There are limited data regarding immunological correlates of protection for the modified vaccinia Ankara (MVA) smallpox vaccine. METHODS: A total of 523 vaccinia-naive subjects were randomized to receive 2 vaccine doses, as lyophilized MVA given subcutaneously, liquid MVA given subcutaneously (liquid-SC group), or liquid MVA given intradermally (liquid-ID group) 28 days apart. For a subset of subjects, antibody-dependent cellular cytotoxicity (ADCC), interferon-γ release enzyme-linked immunospot (ELISPOT), and protein microarray antibody-binding assays were conducted. Protein microarray responses were assessed for correlations with plaque reduction neutralization titer (PRNT), enzyme-linked immunosorbent assay, ADCC, and ELISPOT results. RESULTS: MVA elicited significant microarray antibody responses to 15 of 224 antigens, mostly virion membrane proteins, at day 28 or 42, particularly WR113/D8L and WR101H3L. In the liquid-SC group, responses to 9 antigens, including WR113/D8L and WR101/H3L, correlated with PRNT results. Three were correlated in the liquid-ID group. No significant correlations were observed with ELISPOT responses. In the liquid-ID group, WR052/F13L, a membrane glycoprotein, correlated with ADCC responses. CONCLUSIONS: MVA elicited antibodies to 15 vaccinia strain antigens representing virion membrane. Antibody responses to 2 proteins strongly increased and significantly correlated with increases in PRNT. Responses to these proteins are potential correlates of protection and may serve as immunogens for future vaccine development. CLINICAL TRIALS REGISTRATION: NCT00914732.

RP-REP Ribosomal Profiling Reports: an open-source cloud-enabled framework for reproducible ribosomal profiling data processing, analysis, and result reporting.

Ribosomal profiling is an emerging experimental technology to measure protein synthesis by sequencing short mRNA fragments undergoing translation in ribosomes. Applied on the genome wide scale, this is a powerful tool to profile global protein synthesis within cell populations of interest. Such information can be utilized for biomarker discovery and detection of treatment-responsive genes. However, analysis of ribosomal profiling data requires careful preprocessing to reduce the impact of artifacts and dedicated statistical methods for visualizing and modeling the high-dimensional discrete read count data. Here we present Ribosomal Profiling Reports (RP-REP), a new open-source cloud-enabled software that allows users to execute start-to-end gene-level ribosomal profiling and RNA-Seq analysis on a pre-configured Amazon Virtual Machine Image (AMI) hosted on AWS or on the user's own Ubuntu Linux server. The software works with FASTQ files stored locally, on AWS S3, or at the Sequence Read Archive (SRA). RP-REP automatically executes a series of customizable steps including filtering of contaminant RNA, enrichment of true ribosomal footprints, reference alignment and gene translation quantification, gene body coverage, CRAM compression, reference alignment QC, data normalization, multivariate data visualization, identification of differentially translated genes, and generation of heatmaps, co-translated gene clusters, enriched pathways, and other custom visualizations. RP-REP provides functionality to contrast RNA-SEQ and ribosomal profiling results, and calculates translational efficiency per gene. The software outputs a PDF report and publication-ready table and figure files. As a use case, we provide RP-REP results for a dengue virus study that tested cytosol and endoplasmic reticulum cellular fractions of human Huh7 cells pre-infection and at 6 h, 12 h, 24 h, and 40 h post-infection. Case study results, Ubuntu installation scripts, and the most recent RP-REP source code are accessible at GitHub. The cloud-ready AMI is available at AWS (AMI ID: RPREP RSEQREP (Ribosome Profiling and RNA-Seq Reports) v2.1 (ami-00b92f52d763145d3)).

Transcriptomic and Metabolic Responses to a Live-Attenuated Francisella tularensis Vaccine.

The immune response to live-attenuated Francisella tularensis vaccine and its host evasion mechanisms are incompletely understood. Using RNA-Seq and LC-MS on samples collected pre-vaccination and at days 1, 2, 7, and 14 post-vaccination, we identified differentially expressed genes in PBMCs, metabolites in serum, enriched pathways, and metabolites that correlated with T cell and B cell responses, or gene expression modules. While an early activation of interferon α/ß signaling was observed, several innate immune signaling pathways including TLR, TNF, NF-κB, and NOD-like receptor signaling and key inflammatory cytokines such as Il-1α, Il-1ß, and TNF typically activated following infection were suppressed. The NF-κB pathway was the most impacted and the likely route of attack. Plasma cells, immunoglobulin, and B cell signatures were evident by day 7. MHC I antigen presentation was more actively up-regulated first followed by MHC II which coincided with the emergence of humoral immune signatures. Metabolomics analysis showed that glycolysis and TCA cycle-related metabolites were perturbed including a decline in pyruvate. Correlation networks that provide hypotheses on the interplay between changes in innate immune, T cell, and B cell gene expression signatures and metabolites are provided. Results demonstrate the utility of transcriptomics and metabolomics for better understanding molecular mechanisms of vaccine response and potential host-pathogen interactions.

Proteomic Analysis of Human Immune Responses to Live-Attenuated Tularemia Vaccine.

Francisella tularensis (F. tularensis) is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. F. tularensis can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%. To study the host responses to a live-attenuated tularemia vaccine, peripheral blood mononuclear cell (PBMC) samples were assayed from 10 subjects collected pre- and post-vaccination, using both the 2D-DIGE/MALDI-MS/MS and LC-MS/MS approaches. Protein expression related to antigen processing and presentation, inflammation (PPARγ nuclear receptor), phagocytosis, and gram-negative bacterial infection was enriched at Day 7 and/or Day 14. Protein candidates that could be used to predict human immune responses were identified by evaluating the correlation between proteome changes and humoral and cellular immune responses. Consistent with the proteomics data, parallel transcriptomics data showed that MHC class I and class II-related signals important for protein processing and antigen presentation were up-regulated, further confirming the proteomic results. These findings provide new biological insights that can be built upon in future clinical studies, using live attenuated strains as immunogens, including their potential use as surrogates of protection.

Alterations in the Human Plasma Lipidome in Response to Tularemia Vaccination.

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis. To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids; several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

Plasmablast, Memory B Cell, CD4+ T Cell, and Circulating Follicular Helper T Cell Responses to a Non-Replicating Modified Vaccinia Ankara Vaccine.

Background: Vaccinia is known to induce antibody and cellular responses. Plasmablast, circulating follicular helper T (cTFH) cells, cytokine-expressing CD4 T cells, and memory B cells were compared between subcutaneous (SC) and needle-free jet injection (JI) recipients of non-replicating modified vaccinia Ankara (MVA) vaccine. Methods: Vaccinia-naïve adults received MVA SC or by JI on Days 1 and 29. Vaccinia-specific antibodies were quantified by plaque reduction neutralization test (PRNT) and enzyme-linked immunosorbent assay. Plasmablast, cTFH, and cytokine-expressing CD4 T cells were assessed on Days 1, 8, 15, 29, 36, 43 (cTFH and CD4+ only) and 57. Memory B cells were measured on Days 1 and 57. Results: Of the 36 enrolled subjects, only 22 received both vaccinations and had evaluable specimens after the second vaccine. Plasmablasts peaked one week after each vaccine. Day 15 plasmablasts correlated with peak PRNT titers. cTFH peaked on Days 8 and 36 and correlated with Day 36 plasmablasts. CD4+ peaked at Day 29 and one-third produced ≥2 cytokines. Day 57 memory B cells ranged from 0.1% to 0.17% of IgG-secreting B cells. Conclusions: This study provides insights into the cellular responses to non-replicating MVA, currently used as a vector for a variety of novel vaccines.

Flagellin adjuvanted F1/V subunit plague vaccine induces T cell and functional antibody responses with unique gene signatures.

Yersinia pestis, the cause of plague, could be weaponized. Unfortunately, development of new vaccines is limited by lack of correlates of protection. We used pre- and post-vaccination sera and peripheral blood mononuclear cells from a flagellin adjuvanted F1/V vaccine trial to evaluate for protective markers. Here, we report for the first time in humans that inverse caspase-3 levels, which are measures of protective antibody, significantly increased by 29% and 75% on days 14 and 28 post-second vaccination, respectively. In addition, there were significant increases in T-cell responses on day 28 post-second vaccination. The strongest positive and negative correlations between protective antibody levels and gene expression signatures were identified for IFNG and ENSG00000225107 genes, respectively. Flagellin/F1/V subunit vaccine induced macrophage-protective antibody and significant CD4+ T-cell responses. Several genes associated with these responses were identified that could serve as potential correlates of protection.

Cytomegalovirus Genetic Diversity Following Primary Infection.

BACKGROUND: Infection with multiple cytomegalovirus (CMV) strains (mixed infection) was reported in a variety of hosts. As the virus genetic diversity in primary CMV infection and the changes over time remain incompletely defined, we examined CMV diversity and changes in diversity over time in healthy adolescent females who participated in a phase 2 CMV gB/MF59 vaccine trial. METHODS: CMV genetic diversity was determined by genotyping of 5 genes-gB (UL55), gH (UL75), gN (UL73), US28, and UL144-in urine, saliva, and plasma samples from 15 study subjects. RESULTS: At the time of primary infection, 5 of 12 (42%) urine samples had multiple virus strains, and 50% of vaccine recipients were infected with gB1 genotype (vaccine strain). Mixed infection was documented in all 15 subjects within 3 months after primary infection, and the majority had different CMV genotypes in different compartments. Changes in genotypes over time were observed in all subjects. CONCLUSIONS: Infection with multiple CMV genotypes was common during primary infection and further diversification occurred over time. Infection with gB1 genotype in vaccine recipients suggests a lack of strain-specific protection from the vaccine. As only 5 polymorphic genes were assessed, this study likely underestimated the true genetic diversity in primary CMV infection.

Systems Vaccinology for a Live Attenuated Tularemia Vaccine Reveals Unique Transcriptional Signatures That Predict Humoral and Cellular Immune Responses.

Background: Tularemia is a potential biological weapon due to its high infectivity and ease of dissemination. This study aimed to characterize the innate and adaptive responses induced by two different lots of a live attenuated tularemia vaccine and compare them to other well-characterized viral vaccine immune responses. Methods: Microarray analyses were performed on human peripheral blood mononuclear cells (PBMCs) to determine changes in transcriptional activity that correlated with changes detected by cellular phenotyping, cytokine signaling, and serological assays. Transcriptional profiles after tularemia vaccination were compared with yellow fever [YF-17D], inactivated [TIV], and live attenuated [LAIV] influenza. Results: Tularemia vaccine lots produced strong innate immune responses by Day 2 after vaccination, with an increase in monocytes, NK cells, and cytokine signaling. T cell responses peaked at Day 14. Changes in gene expression, including upregulation of STAT1, GBP1, and IFIT2, predicted tularemia-specific antibody responses. Changes in CCL20 expression positively correlated with peak CD8+ T cell responses, but negatively correlated with peak CD4+ T cell activation. Tularemia vaccines elicited gene expression signatures similar to other replicating vaccines, inducing early upregulation of interferon-inducible genes. Conclusions: A systems vaccinology approach identified that tularemia vaccines induce a strong innate immune response early after vaccination, similar to the response seen after well-studied viral vaccines, and produce unique transcriptional signatures that are strongly correlated to the induction of T cell and antibody responses.

AS03-Adjuvanted H5N1 Avian Influenza Vaccine Modulates Early Innate Immune Signatures in Human Peripheral Blood Mononuclear Cells.

BACKGROUND: Adjuvant System 03 (AS03) markedly enhances responses to influenza A/H5N1 vaccines, but the mechanisms of this enhancement are incompletely understood. METHODS: Using ribonucleic acid sequencing on peripheral blood mononuclear cells (PBMCs) from AS03-adjuvanted and unadjuvanted inactivated H5N1 vaccine recipients, we identified differentially expressed genes, enriched pathways, and genes that correlated with serologic responses. We compared bulk PBMC findings with our previously published assessments of flow-sorted immune cell types. RESULTS: AS03-adjuvanted vaccine induced the strongest differential signals on day 1 postvaccination, activating multiple innate immune pathways including interferon and JAK-STAT signaling, Fcγ receptor (FcγR)-mediated phagocytosis, and antigen processing and presentation. Changes in signal transduction and immunoglobulin genes predicted peak hemagglutinin inhibition (HAI) titers. Compared with individual immune cell types, activated PBMC genes and pathways were most similar to innate immune cells. However, several pathways were unique to PBMCs, and several pathways identified in individual cell types were absent in PBMCs. CONCLUSIONS: Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early activation of innate immune signaling, including a 5- to 8-fold upregulation of FcγR1A/1B/1C genes. Several early gene responses were correlated with HAI titer, indicating links with the adaptive immune response. Although PBMCs and cell-specific results shared key innate immune signals, unique signals were identified by both approaches.

Molecular Evolution of Herpes Simplex Virus 2 Complete Genomes: Comparison between Primary and Recurrent Infections.

Herpes simplex virus 1 (HSV-1) and HSV-2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of recurrent disease over time is not known. We determined the genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped, and amino acid variants resulting from nonsynonymous changes were analyzed based on the secondary and tertiary structures of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage-number clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported here would be a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates.IMPORTANCE The extent to which the HSV-2 genome evolves during multiple episodes of reactivation from its latent state within an infected individual is not known. We used next-generation sequencing techniques to determine whole-genome sequences of four viral samples from two subjects in the Herpevac Trial. The sequence of each subject's well-documented primary isolate was compared with the sequence of the isolate from their fifth or sixth episode of recurrent disease. Only 19 genetic polymorphisms unique to the primary or recurrent isolate were identified, 10 in subject A and 9 in subject B. These observations indicate remarkable genetic conservation between primary and recurrent episodes of HSV-2 infection and imply that strong selection pressures exist to maintain the fidelity of the viral genome during repeated reactivations from its latent state. The genome conservation observed also has implications for the potential success of a therapeutic vaccine.