The Majority of SARS-CoV-2 Plasma Cells are Excluded from the Bone Marrow Long-Lived Compartment 33 Months after mRNA Vaccination.

The goal of any vaccine is to induce long-lived plasma cells (LLPC) to provide life-long protection. Natural infection by influenza, measles, or mumps viruses generates bone marrow (BM) LLPC similar to tetanus vaccination which affords safeguards for decades. Although the SARS-CoV-2 mRNA vaccines protect from severe disease, the serologic half-life is short-lived even though SARS-CoV-2-specific plasma cells can be found in the BM. To better understand this paradox, we enrolled 19 healthy adults at 1.5-33 months after SARS-CoV-2 mRNA vaccine and measured influenza-, tetanus-, or SARS-CoV-2-specific antibody secreting cells (ASC) in LLPC (CD19 - ) and non-LLPC (CD19 + ) subsets within the BM. All individuals had IgG ASC specific for influenza, tetanus, and SARS-CoV-2 in at least one BM ASC compartment. However, only influenza- and tetanus-specific ASC were readily detected in the LLPC whereas SARS-CoV-2 specificities were mostly excluded. The ratios of non-LLPC:LLPC for influenza, tetanus, and SARS-CoV-2 were 0.61, 0.44, and 29.07, respectively. Even in five patients with known PCR-proven history of infection and vaccination, SARS-CoV-2-specific ASC were mostly excluded from the LLPC. These specificities were further validated by using multiplex bead binding assays of secreted antibodies in the supernatants of cultured ASC. Similarly, the IgG ratios of non-LLPC:LLPC for influenza, tetanus, and SARS-CoV-2 were 0.66, 0.44, and 23.26, respectively. In all, our studies demonstrate that rapid waning of serum antibodies is accounted for by the inability of mRNA vaccines to induce BM LLPC.

Circulating antibody-secreting cells are a biomarker for early diagnosis in patients with Lyme disease.

BACKGROUND: Diagnostic immunoassays for Lyme disease have several limitations including: 1) not all patients seroconvert; 2) seroconversion occurs later than symptom onset; and 3) serum antibody levels remain elevated long after resolution of the infection. INTRODUCTION: MENSA (Medium Enriched for Newly Synthesized Antibodies) is a novel diagnostic fluid that contains antibodies produced in vitro by circulating antibody-secreting cells (ASC). It enables measurement of the active humoral immune response. METHODS: In this observational, case-control study, we developed the MicroB-plex Anti-C6/Anti-pepC10 Immunoassay to measure antibodies specific for the Borrelia burgdorferi peptide antigens C6 and pepC10 and validated it using a CDC serum sample collection. Then we examined serum and MENSA samples from 36 uninfected Control subjects and 12 Newly Diagnosed Lyme Disease Patients. RESULTS: Among the CDC samples, antibodies against C6 and/or pepC10 were detected in all seropositive Lyme patients (8/8), but not in sera from seronegative patients or healthy controls (0/24). Serum antibodies against C6 and pepC10 were detected in one of 36 uninfected control subjects (1/36); none were detected in the corresponding MENSA samples (0/36). In samples from newly diagnosed patients, serum antibodies identified 8/12 patients; MENSA antibodies also detected 8/12 patients. The two measures agreed on six positive individuals and differed on four others. In combination, the serum and MENSA tests identified 10/12 early Lyme patients. Typically, serum antibodies persisted 80 days or longer while MENSA antibodies declined to baseline within 40 days of successful treatment. DISCUSSION: MENSA-based immunoassays present a promising complement to serum immunoassays for diagnosis and tracking therapeutic success in Lyme infections.

Poor immunogenicity upon SARS-CoV-2 mRNA vaccinations in autoimmune SLE patients is associated with pronounced EF-mediated responses and anti-BAFF/Belimumab treatment.

Novel mRNA vaccines have resulted in a reduced number of SARS-CoV-2 infections and hospitalizations. Yet, there is a paucity of studies regarding their effectiveness on immunocompromised autoimmune subjects. In this study, we enrolled subjects naïve to SARS-CoV-2 infections from two cohorts of healthy donors (HD, n=56) and systemic lupus erythematosus (SLE, n=69). Serological assessments of their circulating antibodies revealed a significant reduction of potency and breadth of neutralization in the SLE group, only partially rescued by a 3rd booster dose. Immunological memory responses in the SLE cohort were characterized by a reduced magnitude of spike-reactive B and T cell responses that were strongly associated with poor seroconversion. Vaccinated SLE subjects were defined by a distinct expansion and persistence of a DN2 spike-reactive memory B cell pool and a contraction of spike-specific memory cTfh cells, contrasting with the sustained germinal center (GC)-driven activity mediated by mRNA vaccination in the healthy population. Among the SLE-associated factors that dampened the vaccine responses, treatment with the monoclonal antibody anti-BAFF/Belimumab (a lupus FDA-approved B cell targeting agent) profoundly affected the vaccine responsiveness by restricting the de novo B cell responses and promoting stronger extra-follicular (EF)-mediated responses that were associated with poor immunogenicity and impaired immunological memory. In summary, this study interrogates antigen-specific responses and characterized the immune cell landscape associated with mRNA vaccination in SLE. The identification of factors associated with reduced vaccine efficacy illustrates the impact of SLE B cell biology on mRNA vaccine responses and provides guidance for the management of boosters and recall vaccinations in SLE patients according to their disease endotype and modality of treatment.

Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID.

While immunologic correlates of COVID-19 have been widely reported, their associations with post-acute sequelae of COVID-19 (PASC) remain less clear. Due to the wide array of PASC presentations, understanding if specific disease features associate with discrete immune processes and therapeutic opportunities is important. Here we profile patients in the recovery phase of COVID-19 via proteomics screening and machine learning to find signatures of ongoing antiviral B cell development, immune-mediated fibrosis, and markers of cell death in PASC patients but not in controls with uncomplicated recovery. Plasma and immune cell profiling further allow the stratification of PASC into inflammatory and non-inflammatory types. Inflammatory PASC, identifiable through a refined set of 12 blood markers, displays evidence of ongoing neutrophil activity, B cell memory alterations, and building autoreactivity more than a year post COVID-19. Our work thus helps refine PASC categorization to aid in both therapeutic targeting and epidemiological investigation of PASC.

Dysregulated naive B cells and de novo autoreactivity in severe COVID-19.

Severe SARS-CoV-2 infection1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic2-5. More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential6-10, although their origins and resolution have remained unclear11. Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity12,13, as a dominant feature of severe and critical COVID-19 (refs. 14-18). Here, using single-cell B cell repertoire analysis of patients with mild and severe disease, we identify the expansion of a naive-derived, low-mutation IgG1 population of antibody-secreting cells (ASCs) reflecting features of low selective pressure. These features correlate with progressive, broad, clinically relevant autoreactivity, particularly directed against nuclear antigens and carbamylated proteins, emerging 10-15 days after the onset of symptoms. Detailed analysis of the low-selection compartment shows a high frequency of clonotypes specific for both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against the glomerular basement membrane. We further identify the contraction of this pathway on recovery, re-establishment of tolerance standards and concomitant loss of acute-derived ASCs irrespective of antigen specificity. However, serological autoreactivity persists in a subset of patients with postacute sequelae, raising important questions as to the contribution of emerging autoreactivity to continuing symptomology on recovery. In summary, this study demonstrates the origins, breadth and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Detection of Newly Secreted Antibodies Predicts Nonrecurrence in Primary Clostridioides difficile Infection.

Within 8 weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no clear clinical correlates or validated biomarkers that can predict recurrence during primary infection. This study demonstrated the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence. Forty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Samples of serum and a novel matrix from circulating plasmablasts called "medium-enriched for newly synthesized antibodies" (MENSA) were collected during weeks 1, 2, and 4. Antibodies specific for 10 C. difficile antigens were measured in each sample. Among the 46 C. difficile-infected patients, 9 (19.5%) experienced recurrence within 8 weeks of primary infection. Among the 37 nonrecurrent patients, 23 (62%; 23/37) had anti-C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred early (within the first 12 days post-symptom onset), including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (51%; 19/37). In contrast, none (0%; 0/9) of the patients who subsequently recurred after hospitalization produced antibodies specific for any of the three C. difficile toxin antigens. Thus, patients with a negative early MENSA response against all three C. difficile toxin antigens had a 19-fold greater relative risk of recurrence. MENSA and serum levels of immunoglobulin A (IgA) and/or IgG antibodies for three C. difficile toxins have prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence thereby providing a biomarker of protection from recurrent CDI. Patients who are positive by this immunoassay are unlikely to suffer a recurrence. Early identification of patients at risk for recurrence by negative MENSA creates opportunities for targeted prophylactic strategies that can reduce the incidence, cost, and morbidity due to recurrent CDI.

Delayed Kinetics of IgG, but Not IgA, Antispike Antibodies in Transplant Recipients following SARS-CoV-2 Infection.

BACKGROUND: Kidney transplant recipients are at increased risk of severe outcomes during COVID-19. Antibodies against the virus are thought to offer protection, but a thorough characterization of anti-SARS-CoV-2 immune globulin isotypes in kidney transplant recipients following SARS-CoV-2 infection has not been reported. METHODS: We performed a cross-sectional study of 49 kidney transplant recipients and 42 immunocompetent controls at early (≤14 days) or late (>14 days) time points after documented SARS-CoV-2 infection. Using a validated semiquantitative Luminex-based multiplex assay, we determined the abundances of IgM, IgG, IgG1-4, and IgA antibodies against five distinct viral epitopes. RESULTS: Kidney transplant recipients showed lower levels of total IgG antitrimeric spike (S), S1, S2, and receptor binding domain (RBD) but not nucleocapsid (NC) at early versus late time points after SARS-CoV-2 infection. Early levels of IgG antispike protein epitopes were also lower than in immunocompetent controls. Anti-SARS-CoV-2 antibodies were predominantly IgG1 and IgG3, with modest class switching to IgG2 or IgG4 in either cohort. Later levels of IgG antispike, S1, S2, RBD, and NC did not significantly differ between cohorts. There was no significant difference in the kinetics of either IgM or IgA antispike, S1, RBD, or S2 on the basis of timing after diagnosis or transplant status. CONCLUSIONS: Kidney transplant recipients mount early anti-SARS-CoV-2 IgA and IgM responses, whereas IgG responses are delayed compared with immunocompetent individuals. These findings might explain the poor outcomes in transplant recipients with COVID-19. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/JASN/2021_11_23_briggsgriffin112321.mp3.

One-Stop Serum Assay Identifies COVID-19 Disease Severity and Vaccination Responses.

SARS-CoV-2 has caused over 100,000,000 cases and almost 2,500,000 deaths globally. Comprehensive assessment of the multifaceted antiviral Ab response is critical for diagnosis, differentiation of severity, and characterization of long-term immunity, especially as COVID-19 vaccines become available. Severe disease is associated with early, massive plasmablast responses. We developed a multiplex immunoassay from serum/plasma of acutely infected and convalescent COVID-19 patients and prepandemic and postpandemic healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 nucleocapsid (N), spike domain 1 (S1), S1-receptor binding domain (RBD) and S1-N-terminal domain. For diagnosis, the combined [IgA + IgG + IgM] or IgG levels measured for N, S1, and S1-RBD yielded area under the curve values ≥0.90. Virus-specific Ig levels were higher in patients with severe/critical compared with mild/moderate infections. A strong prozone effect was observed in sera from severe/critical patients-a possible source of underestimated Ab concentrations in previous studies. Mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared with severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 mo after symptom onset. Measurement of the Ab responses in sera from 18 COVID-19-vaccinated patients revealed specific responses for the S1-RBD Ag and none against the N protein. This highly sensitive, SARS-CoV-2-specific, multiplex immunoassay measures the magnitude, complexity, and kinetics of the Ab response and can distinguish serum Ab responses from natural SARS-CoV-2 infections (mild or severe) and mRNA COVID-19 vaccines.

Novel immunoassay for diagnosis of ongoing Clostridioides difficile infections using serum and medium enriched for newly synthesized antibodies (MENSA).

BACKGROUND: Clostridioides difficile infections (CDI) have been a challenging and increasingly serious concern in recent years. While early and accurate diagnosis is crucial, available assays have frustrating limitations. OBJECTIVE: Develop a simple, blood-based immunoassay to accurately diagnose patients suffering from active CDI. MATERIALS AND METHODS: Uninfected controls (N = 95) and CDI patients (N = 167) were recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. difficile antigens. RESULTS: Sixty-six percent of CDI patients produced measurable responses to C. difficile antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in MENSA, and 27/167 (16%) were detectable only in serum. DISCUSSION: We describe the results of a multiplex immunoassay for the diagnosis of ongoing CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, serum-positive only, MENSA- and serum-positive, and MENSA- and serum-negative. The 30% of patients who were MENSA-positive only may be accounted for by nascent antibody secretion prior to seroconversion. Conversely, the serum-positive only subset may have been more advanced in their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI patients who were not identified using MENSA or serum immunoassays. IMPORTANCE: While there was considerable overlap between patients identified through MENSA and serum, each method detected a distinctive patient group. The combined use of both MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host humoral immune responses in CDI patients.

Relaxed peripheral tolerance drives broad de novo autoreactivity in severe COVID-19.

An emerging feature of COVID-19 is the identification of autoreactivity in patients with severe disease that may contribute to disease pathology, however the origin and resolution of these responses remain unclear. Previously, we identified strong extrafollicular B cell activation as a shared immune response feature between both severe COVID-19 and patients with advanced rheumatic disease. In autoimmune settings, this pathway is associated with relaxed peripheral tolerance in the antibody secreting cell compartment and the generation of de novo autoreactive responses. Investigating these responses in COVID-19, we performed single-cell repertoire analysis on 7 patients with severe disease. In these patients, we identify the expansion of a low-mutation IgG1 fraction of the antibody secreting cell compartment that are not memory derived, display low levels of selective pressure, and are enriched for autoreactivity-prone IGHV4-34 expression. Within this compartment, we identify B cell lineages that display specificity to both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against glomerular basement membrane, and describe progressive, broad, clinically relevant autoreactivity within these patients correlated with disease severity. Importantly, we identify anti-carbamylated protein responses as a common hallmark and candidate biomarker of broken peripheral tolerance in severe COVID-19. Finally, we identify the contraction of this pathway upon recovery, and re-establishment of tolerance standards coupled with a concomitant loss of acute-derived ASCs irrespective of antigen specificity. In total, this study reveals the origins, breadth, and resolution of acute-phase autoreactivity in severe COVID-19, with significant implications in both early interventions and potential treatment of patients with post-COVID sequelae.

Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection.

BACKGROUND: SARS-CoV-2 has caused over 36,000,000 cases and 1,000,000 deaths globally. Comprehensive assessment of the multifaceted anti-viral antibody response is critical for diagnosis, differentiation of severe disease, and characterization of long-term immunity. Initial observations suggest that severe disease is associated with higher antibody levels and greater B cell/plasmablast responses. A multi-antigen immunoassay to define the complex serological landscape and clinical associations is essential. METHODS: We developed a multiplex immunoassay and evaluated serum/plasma from adults with RT-PCR-confirmed SARS-CoV-2 infections during acute illness (N=52) and convalescence (N=69); and pre-pandemic (N=106) and post-pandemic (N=137) healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 Nucleocapsid (N), Spike domain 1 (S1), receptor binding domain (S1-RBD) and S1-N-terminal domain (S1-NTD). RESULTS: To diagnose infection, the combined [IgA+IgG+IgM] or IgG for N, S1, and S1-RBD yielded AUC values -0.90 by ROC curves. From days 6-30 post-symptom onset, the levels of antigen-specific IgG, IgA or [IgA+IgG+IgM] were higher in patients with severe/critical compared to mild/moderate infections. Consistent with excessive concentrations of antibodies, a strong prozone effect was observed in sera from severe/critical patients. Notably, mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared to severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 months. CONCLUSION: This SARS-CoV-2 multiplex immunoassay measures the magnitude, complexity and kinetics of the antibody response against multiple viral antigens. The IgG and combined-isotype SARS-CoV-2 multiplex assay is highly diagnostic of acute and convalescent disease and may prognosticate severity early in illness. ONE SENTENCE SUMMARY: In contrast to patients with moderate infections, those with severe COVID-19 develop prominent, early antibody responses to S1 and N proteins.

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19.

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

The genomic substrate for adaptive radiation in African cichlid fish.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.