We report here the design, synthesis and evaluation of a series of HIV-1 protease inhibitors that incorporate substituted oxaspirocyclic carbamate derivatives to serve as the P2 ligands. Various substituted ligand derivatives were synthesized in a racemic manner, using a tandem Prins/pinacol reaction as the key reaction. This reaction sets the relative stereochemistry of the oxaspirocyclic template in a highly diastereoselective manner. Reaction of the resulting ketone with enantiopure (S)-tert-butyl sulfinamide provided a convenient pathway to resolve the oxaspirocyclic ketone derivatives. The absolute stereochemical identity was determined by X-ray crystallography. The structure-activity studies demonstrate the effect of the stereochemistry of the oxaspirocyclic ring systems as well as the substitution effect on the aromatic ring. Several inhibitors exhibited potent HIV-1 protease inhibitory activity. One of these inhibitors displayed subnanomolar HIV-1 protease affinity and also exhibited potent antiviral activity. A high-resolution X-ray crystal structure of this inhibitor-bound HIV-1 protease show that the oxaspirocyclic P2 ligand forms an unconventional C-H⋯O bond with the backbone carboxyl group of Gly48' and an interesting N-H … π interaction with the aromatic ring in the S2 subsite of HIV-1 protease active site.

Drugs that covalently bind to their target molecules form strong and irreversible interactions with each other. Covalent inhibitors have recently attracted attention because of their enhanced structural selectivity within the target cavity, which results in prolonged interactions, even at low doses. Here, we report that ACAi-001 is a human immunodeficiency virus (HIV)-1 capsid (CA)-targeting compound with covalent binding to CA, estimating long-lasting effects for HIV inhibition. Using a library of millions of compounds, ACAi-001 was identified through in silico screening. This compound is designed to target the hydrophobic cavity in the N-terminal domain of CA and, in a cell-based assay, was found to inhibit HIV-1 replication. ACAi-001 covalently bound to CA and induced aberrant CA multimerization and degradation, as assessed by western blotting, size-exclusion chromatography, thermal stability assay, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry. ACAi-001, which has two putative covalent interactions in its chemical structure, binds directly to serine 16 in the N-terminal domain-targeting cavity and to cysteine 198 or 218 in the C-terminal domain of CA. This unique binding profile of ACAi-001 to CA induces multiple CA dysfunctions such as CA multimerization and degradation, resulting in disruption of the HIV core, thereby inhibiting HIV-1 infection and replication. ACAi-001, which covalently binds to and degrades CA in an unique manner, may reveal new aspects of HIV core assembly and disassembly and is a promising candidate as a next-generation anti-HIV-1 CA inhibitor. Significance Statement Lenacapavir (LEN) is a first-in-class CA inhibitor used clinically to inhibit HIV-1 replication. LEN is a long-acting capsid inhibitor with a convenient dosing regimen and is effective for both treating multidrug-resistant HIV and as pre-exposure prophylaxis. ACAi-001 targets specific CA cavities and covalently binds to CA in a distinct manner from LEN. ACAi-001 forms two covalent interactions with CA, leading to CA multimerization and degradation to cause multiple CA dysfunctions. Therefore, more potent derivatives of ACAi-001 with covalent interactions may serve as long-acting capsid inhibitors. ACAi-001 shows potential as a next-generation HIV-1 capsid inhibitor against HIV-1 infection and can be used to reveal details on HIV core assembly and disassembly in the HIV-1 life cycle. ### Competing Interest Statement The authors have declared no competing interest. Japan Society for the Promotion of Science, https://ror.org/00hhkn466, 23K24379 (22H03120), 18K08435, and 15K09574

We describe here the design, synthesis, and X-ray structural studies of a new class of HIV-1 protease inhibitors containing 8-oxabicyclo[3.2.1]octanol-derived P2 ligands. We investigated the functional effect of these stereochemically defined fused-poly cyclic ligands on enzyme inhibition and antiviral activity in MT-2 cells. The tricyclic core of 8-oxabicyclo[3.2.1]octan-6-ol is designed to interact with the residues in the S2 subsite of HIV-1 protease. The syntheses of the ligands were carried out using the [5+2]-cycloaddition as the key step. Several inhibitors exhibited potent enzyme inhibitory activity. High resolution room-temperature X-ray structures of inhibitor-bound HIV-1 protease were determined. These structures provided important molecular insights for further design and optimization of inhibitor potency.

Coronavirus disease-2019 (COVID-19) remains a critical global health concern. We developed a fully automated, high-throughput competition immunoassay to elucidate how epitope recognition on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) correlates with neutralizing activity. Analysis of clinical samples from both SARS-CoV-2-infected and vaccinated individuals revealed that vaccination elicits significantly higher antibody titers across multiple S1 subunit epitopes compared to natural infection. Notably, median antibody levels against the receptor-binding motif (RBM) exceeded 50% in both cohorts, highlighting the RBM as a key target for antibody induction irrespective of immune origin. Furthermore, the strongest correlation with neutralizing activity was observed for antibodies directed against the broader S1 subunit, indicating that epitopes outside the RBM also contribute to neutralization. These findings underscore the importance of both RBM- and non-RBM-directed antibodies in effective immune defense against SARS-CoV-2. Our assay enables large-scale, reliable quantification of neutralizing antibodies and provides critical insights for developing improved diagnostic antigens and vaccine strategies aimed at eliciting robust, multi-epitope immune responses.

Substituted tetrahydrofuran derivatives were designed and synthesized to serve as the P2 ligand for a series of potent HIV-1 protease inhibitors. Both enantiomers of the tetrahydrofuran derivatives were synthesized stereoselectivity in optically active forms using lipase-PS catalyzed enzymatic resolution as the key step. These tetrahydrofuran derivatives are designed to promote hydrogen bonding and van der Waals interactions with the backbone atoms in the S2 subsite of the HIV-1 protease active site. Several inhibitors displayed very potent HIV-1 protease inhibitory activity. A high-resolution X-ray crystal structure of an inhibitor-bound HIV-1 protease provided important insight into the ligand binding site interactions in the active site.

We read with interest the article reported by Pozo-Balado et al about humoral responses post 2nd/3rd doses of SARS-CoV-2 mRNA vaccination (BNT162b2) in elder individuals,1Pozo-Balado M.D.M. Bulnes-Ramos Á. Garrido-Rodríguez V. Olivas-Martínez I. Lozano C. González-Escribano M.F. et al.Longitudinal age differences in humoral responses to the COVID-19 vaccine in the elderly are lost after the third dose.J Infect. 2022; S0163–4453 (00692-2)Google Scholar and we have recently reported the protective effect after 4th dose of BNT162b2 vaccination in the cohort study including participants older than 60 years and those with risk factors.2Amano M. Otsu S. Ichikawa Y. Higashi-Kuwata N. Matsushita S. Shimada S. et al.Restoration of neutralization activity against Omicrons BA.2 and BA.5 in older adults and individuals with risk factors following the 4th-dose of SARS-CoV-2 BNT162b2 vaccine.J Infect Dis. 2022; 227: 161-163Crossref PubMed Scopus (5) Google Scholar Here, we examined the effects of heterologous (1st-3rd/BNT162b2 and 4th/mRNA-1273) SARS-CoV-2 vaccinations and consecutive 4-times (homologous) BNT162b2 vaccinations on neutralizing activity in sera from risk-free health care workers (HCWs) against SARS-CoV-2 Wuhan strain and Omicron BA.5 sublineages, and analyzed longitudinal changes of neutralization activity pre- and post-2nd∼4th BNT162b2 doses over 550 days. In the present prospective clinical study, 104 of 225 HCWs in Kumamoto General Hospital, Japan (225 were initially recruited in the primary clinical study3Maeda K. Amano M. Uemura Y. Tsuchiya K. Matsushima T. Noda K. et al.Correlates of neutralizing/SARS-CoV-2-S1- binding antibody response with adverse effects and immune kinetics in BNT162b2- vaccinated individuals.Sci Rep. 2021; 11: 22848Crossref PubMed Scopus (38) Google Scholar), who were younger than 60 years of age and free from pre-existing diseases/risk factors (see demographic characteristics in Supplementary Table 1A), were enrolled. Out of 106, 33 participants received heterologous (1st-3rd/BNT162b2 [Pfizer/BioNTech], 4th/mRNA-1273 [Moderna]) SARS-CoV-2 vaccinations, while 71 of 106 participants received consecutive (homologous) 4 doses of BNT162b2 vaccinations. The SARS-CoV-2 neutralizing activity (NT50) of their sera against the Wuhan strain of SARS-CoV-2 (SARS-CoV-2Wuhan) was determined over 550 days using sera collected consecutively on (i) 1-week post 1st dose, (ii) 1-week post 2nd dose, (iii) 1-week pre 3rd dose, (iv) 2-weeks post 3rd dose, (v) 1-week pre 4th dose, and (vi) 2-weeks post 3rd dose (see Supplementary Methods), as an extension of our previous reports.2Amano M. Otsu S. Ichikawa Y. Higashi-Kuwata N. Matsushita S. Shimada S. et al.Restoration of neutralization activity against Omicrons BA.2 and BA.5 in older adults and individuals with risk factors following the 4th-dose of SARS-CoV-2 BNT162b2 vaccine.J Infect Dis. 2022; 227: 161-163Crossref PubMed Scopus (5) Google Scholar, 3Maeda K. Amano M. Uemura Y. Tsuchiya K. Matsushima T. Noda K. et al.Correlates of neutralizing/SARS-CoV-2-S1- binding antibody response with adverse effects and immune kinetics in BNT162b2- vaccinated individuals.Sci Rep. 2021; 11: 22848Crossref PubMed Scopus (38) Google Scholar, 4Amano M. Maeda K. Tsuchiya K. Shimada S. Mitsuya H. Third-dose BNT162b2 vaccination elicits markedly high-level SARS-CoV-2-neutralizing antibodies in vaccinees who poorly responded to second dose in Japan.J Infect Dis. 2022; 226: 2038-2039Crossref PubMed Scopus (6) Google Scholar, 5Amano M. Otsu S. Maeda K. Uemura Y. Shimizu Y. Omata K. et al.Neutralization activity of Sera/IgG Preparations from fully BNT162b2 vaccinated individuals against SARS-CoV-2 Alpha Beta Gamma Delta and Kappa variants.Sci Rep. 2022; 12: 13524Crossref PubMed Scopus (8) Google Scholar We also evaluated the profile of S1-binding IgG in sera following pre/post 3rd and 4th dose (on days-280/300 and −530/550 from an initial dose). In addition, we determined NT50s of the same sera using VeroE6TMPRSS cells against infectious Omicron BA.5 sublineages, whose emergence has been associated with the present explosive in new COVID-19 cases globally.6World Health Organization. COVID-19 Weekly Epidemiological Update. 2022. 〈https://www.who.int/publications/m/item/weekly-epidemiological-update-on-covid-19---16-november-2022〉 (Accessed date: 27-Nov.2022).Google Scholar Neutralizing activity of sera against SARS-CoV-2Wuhan was seen elevated moderately on day-28 (1-week post 2nd dose) samples in both groups (Fig. 1A, geometric mean [gMean]-NT50 = 387–447), while remarkable elevations were observed in neutralizing activity in the same participants’ sera of day-300 (2-weeks post 3rd-dose administration), achieving gMean-NT50 of 2567–2534, and the values were equivalent between both groups (Fig. 1A). On day-530 (1-week pre 4th dose), the gMean-NT50 remarkable decreased down to 260–249, around 10% of the peak value of day 300 (2 weeks post 3rd dose) (Fig. 1A). However, by day 550 (2 weeks post 4th dose), neutralization activity was restored to 1790 in the heterologous group and 2039 in the homologous group (Fig. 1A). The gMean values of S1-binding IgG in pre/post 4th-dose sera also showed increase from 125 on day 530 (1-week pre 4th dose) to 929 on day 550 (2 weeks post 4th dose) in the heterologous group and 108 on days 530–893 on day 550 in the homologous group (Supplementary Table 1B). When we evaluated neutralization activity in sera from both groups using infectious Omicron variants, gMean-NT50 values against BA.5 on day 550 (2 weeks post 4th dose) sera were 130 in the heterologous group (ranges;<20–680) and 120 in the homologous group (ranges; 36–2424), 92.7% and 94.1% reductions were observed compared to those, respectively, against Wuhan strain (Fig. 1B). Also, against BA.2, gMean-NT50 values of day-550 sera were 216 in the heterologous group (ranges; 42–989) and 213 in the homologous group (ranges; 64–2223), 87.9% and 89.5% reductions were observed compared to those against Wuhan strain, respectively (Fig. 1B). In the present cohort study, the neutralization activity elicited by the heterologous SARS-CoV-2 vaccinations was moderately weaker (but not statistically significant; p = 0.055) as compared to consecutive 4 doses of BNT162b2 (homologous) vaccinations in risk-free HCWs. Compared to the significantly boosted response elicited by 3rd dose of BNT162b2, the magnitudes of neutralizing activity against SARS-CoV-2Wuhan after 4th dose were not greater than those after 3rd dose in both groups, similar to the results we have previously reported in the cohort study of participants with risk factors.2Amano M. Otsu S. Ichikawa Y. Higashi-Kuwata N. Matsushita S. Shimada S. et al.Restoration of neutralization activity against Omicrons BA.2 and BA.5 in older adults and individuals with risk factors following the 4th-dose of SARS-CoV-2 BNT162b2 vaccine.J Infect Dis. 2022; 227: 161-163Crossref PubMed Scopus (5) Google Scholar Against Omicrons/BA.2 and BA.5, post 4th dose sera of both groups showed significantly reduced neutralization activity, suggesting that despite the 4 doses of Wuhan-strain-based mRNA vaccination, breakthrough infections are likely to continue to occur. It remains to be determined to what extent the Omicron-specific bivalent vaccines strengthen Omicron-specific protection in risk-free individuals who have already acquired 4 regular doses of Wuhan-strain-based vaccine. This research was supported in part by a Grant from the Japan Agency for Medical Research and Development (AMED) to H. Mitsuya (Grant Number 20fk0108502), in part by a grant for COVID-19 to H. Mitsuya (Grant Number 19A3001) from the Intramural Research Program of National Center for Global Health and Medicine, and in part by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, US (H. Mitsuya).

Hepatitis B is a viral hepatitis, which is caused by infection of hepatitis B virus (HBV). This disease progresses to chronic hepatitis, cirrhosis and liver cancer. To treat hepatitis B, exclusion of virus and covalently closed circular DNA (cccDNA) that is formed in hepatocyte nucleus is necessary. A hepatitis B capsid protein (HBc) is an indispensable protein, which forms the capsid that encapsulates viral DNA. Since HBc is correlated to the transcriptional regulation of cccDNA, this protein would be an attractive target for complete cure of hepatitis B. By in silico screening of a library of compounds, a small compound, Cpd4 (1), which binds to a hydrophobic cavity located in the inner pocket on the tetramer interface of HBc proteins, was identified. In anti-HBV assays, this synthetic compound, Cpd4 (1) decreased the amount of HBV core related antigen (HBcrAg), which has been correlated with the proliferation of HBV, and decreased the amount of HBV surface antigen (HBsAg), which is correlated with the amount of cccDNA. Based on Cpd4 (1) as a lead compound, 20 derivatives of 1 were designed and synthesized and their structure-activity relationships were examined. As a result, specific interactions between each compound and amino acid residues of the target protein appeared to be unimportant but the shape/size of compounds which can bind to the hydrophobic cavity might be important in the expression of high anti-HBV activity, and a more potent derivative, TKB-HBV-CA-001 (3b), was discovered. These results will be useful in the development of novel anti-HBV agents for a complete cure of hepatitis B.

Structure-based design, synthesis, X-ray structural studies, and biological evaluation of a new series of potent HIV-1 protease inhibitors are described. These inhibitors contain various pyridyl-pyrimidine, aryl thiazole or alkylthiazole derivatives as the P2 ligands in combination with darunavir-like hydroxyethylamine sulfonamide isosteres. These heterocyclic ligands are inherent to kinase inhibitor drugs, such as nilotinib and imatinib. These ligands are designed to make hydrogen bonding interactions with the backbone atoms in the S2 subsite of HIV-1 protease. Various benzoic acid derivatives have been synthesized and incorporation of these ligands provided potent inhibitors that exhibited subnanomolar level protease inhibitory activity and low nanomolar level antiviral activity. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease were determined. These structures provided important ligand-binding site interactions for further optimization of this class of protease inhibitors.

SARS-CoV-2-BA.4/5-adapted-bivalent-BNT162b2-vaccine (bvBNT), developed in response to the recent emergence of immune-evasive Omicron-variants, has been given to individuals who completed at least 2-doses of the monovalent-BNT162b2-vaccine (mvBNT). In the present cohort study, we evaluated neutralization-titers (NT50s) against Wuhan-strain (SCoV2Wuhan) and Omicron-sublineages including BA.2/BA.5/BQ.1.1/XBB/XBB.1.5, and vaccine-elicited S1-binding-IgG in sera from participants-vaccinated with 5th-bvBNT following 4th-mvBNT. The 5th-bvBNT-dose elicited good protective-activity against SCoV2Wuhan with geometric-mean (gMean)-NT50 of 1966–2091, higher than the peak-values post-4th-mvBNT with no statistical significance, and favorable neutralization-activity against not only BA.5 but also BA.2, with ~ 3.2-/~ 2.2-fold greater gMean-NT50 compared to the peak-values post-4th-mvBNT-dose, in participants with or without risk factors. However, neutralization-activity of sera post-5th-bvBNT-dose was low against BQ.1.1/XBB/XBB.1.5. Interestingly, participants receiving bvBNT following breakthrough (BT) infection during Omicron-wave had significantly enhanced neutralization-activity against SCoV2Wuhan/BA.2/BA.5 with ~ 4.6-/~ 6.3-/~ 8.1-fold greater gMean-NT50, respectively, compared to uninfected participants receiving bvBNT. Sera from BT-infected-participants receiving bvBNT had enhanced neutralization-activity against BQ.1.1/XBB/XBB.1.5 by ~ 3.8-fold compared to those from the same participants post-4th-mvBNT-dose, and had enhanced gMean-NT50 ~ 5.4-fold greater compared to those of uninfected-participants’ sera post-bvBNT. These results suggest that repeated stimulation brought about by exposure to BA.5’s-Spike elicit favorable cross-neutralization-activity against various SARS-CoV-2-variants.

We report here the synthesis and biological evaluation of darunavir derived HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. The P2 ' 4-amino functionality was modified to make a number of amide derivatives to interact with residues in the S2 ' subsite of the HIV-1 protease active site. Several compounds exhibited picomolar enzyme inhibitory and low nanomolar antiviral activity. The X-ray crystal structure of the chloroacetate derivative bound to HIV-1 protease was determined. Interestingly, the active chloroacetate group converted to the acetate functionality during X-ray exposure. The structure revealed that the P2 ' carboxamide functionality makes enhanced hydrogen bonding interactions with the backbone atoms in the S2 '-subsite.

In the present prospective study, 225 individuals in Kumamoto General Hospital, Japan, who received two-doses of BNT162b2 vaccine were enrolled/followed up over 150 days and neutralizing activity (NT 50 ) of their sera and antiviral activity (EC 50 ) of IgG purified from sera on day-60 post-1st-dose were determined against wild-type SARS-CoV-2 (SARS-CoV-2 Wuhan ) (n = 211) and 9 variants (Alpha, Beta, Gamma, Delta, and Kappa) (n = 45). Time-dependent changes of IgG-activity (n = 25) against SARS-CoV-2 Wuhan and variants were also examined. Day-60 sera showed reduced NT 50 by more than 50% against all variants examined, and greatest reduction was seen with Beta. IgG fractions of high-responders and moderate-responders showed similar fold-changes in EC 50 against each variant compared to SARS-CoV-2 Wuhan . Evaluation of EC 50 of IgG obtained at different time-points (day-28 to -150) revealed time-dependent reduction of activity against all variants. However, against Delta, relatively long-lasting favorable antiviral activity (at least 150 days) was observed. Our data strongly suggest that the successful antecedent scale-up of mRNA-based vaccine administrations in Japan was the primary contributor to the lessening of the otherwise more devastating SARS-CoV-2 pandemic wave caused by the Delta variant. The present data that the effectiveness of vaccine against the then-dominant SARS-CoV-2 variant was likely associated with the moderation of the COVID-19 pandemic wave suggest that as in the case of influenza vaccines, the development of multivalent mRNA-based vaccines represent a generalizable approach to pre-emptively respond pandemic with mutable pathogens.

Abstract In the present prospective study, 225 individuals in Kumamoto General Hospital, Japan, who received two-doses of BNT162b2 vaccine were enrolled/followed up over 150 days. Neutralizing activity (NT50) of sera on day-60 post-1st -dose against wild-type (SARS-CoV-2Wuhan)(n = 211) and 9 variants (VOCs; Alpha/Beta/Gamma/Delta, and VUM; Kappa)(n = 45) were examined. Also, antiviral activity (EC50) of IgG obtained from day-60 sera against SARS-CoV-2Wuhan and 9 variants were evaluated (n = 45). Finally, time-dependent alterations of IgG-activity (n = 25) against SARS-CoV-2Wuhan/variants were examined. Day-60 sera showed reduced NT50 by more than 50% against all variants, and greatest reduction was seen with Beta. IgG of high-responders (IgGhighs) and moderate-responders (IgGmoderates) showed similar fold-changes in EC50 against each variant compared to SARS-CoV-2Wuhan. However, absolute EC50 of IgGhighs were 2 ~ 3-folds greater than those of IgGmoderates against all SARS-CoV-2s. Evaluation of EC50 of IgG obtained at different time-points (day-28 to -150) revealed time-dependent reduction of IgG-activity against various VOCs/VUM. However, against Delta, relatively long-lasting favorable EC50 (at least 150 days) of both IgG were observed. Our data strongly suggest that rapid expansion of vaccine administration in Japan was remarkably effective to repress SARS-CoV-2 pandemic with the spread of Delta, and give insights in better understanding of the antiviral-efficacy of SARS-CoV-2 vaccine and vaccine-elicited immune-response.

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2 ' ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2 ' ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.

A variety of unsaturated selenoesters (including phenolic ones) were produced in good to high yields and with high E/Z ratios using TiCl4-promoted aldol condensation between Se-phenyl selenoacetate and their respective aldehydes without aqueous workup. A representative phenolic unsaturated selenoester was applied to acylation of tyrosine methyl ester without protection of the phenolic hydroxy groups to furnish the corresponding amino acid conjugate. The conjugate reduction of the unsaturated selenoesters including phenolic ones and selenocoumarin with HSiEt3 was catalyzed by B(C6F5)3 to afford the corresponding saturated selenoesters in good to high yields. This method was also applicable to the reduction of a saturated selenoester to the corresponding O-silyl hemiselenoacetal in a high yield. Moreover, most acyclic unsaturated selenoesters were found to show good multiple antiviral activities against HIV-1, HBV, and SARS-CoV-2.

TO THE EDITOR—We read with great interest the article by Saciuk et al demonstrating that, in a retrospective cohort study in Israel, an additional dose of BNT162b2 vaccine 6 months after initial 2-dose vaccination bolsters protection against infection, with a vaccine effectiveness of 89%, as assessed during August–October 2021, when the majority of infections were due to the Delta variant [1]. Recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) studies using pseudovirus have shown that a third dose of BNT162b2 well elicits neutralizing antibodies against VOC (variants of concern) including Omicron (B.1.1.529, BA.1) [2–4]. However, Cheng et al very recently reported a single-source outbreak of Omicron BA.2 sublineage in Hong Kong, which indicates high transmissibility of Omicron/BA.2 [5] and poses further concerns on the efficacy of anti– SARS-CoV-2 vaccines. In the present prospective study, enrolling 225 health care workers (see demographic characteristics in Supplementary Table 1), who received 3 doses of BNT162b2 in Japan, we consecutively determined SARS-CoV-2 neutralizing activity (50% neutralizing titer, NT50) of their sera using VeroE6 cells and its kinetics/profiles over 300 days following the first dose; this is a continuation of our previous study [6]. We also determined NT50 of selected sera using VeroE6 and HeLa cells against infectious VOC, including Delta and Omicrons (BA.1 and BA.2), whose emergence has been associated with a steep increase in coronavirus disease 2019 (COVID-19) cases and hospitalizations (experimental details are provided in the “Methods” section of Supplementary Materials). There was significant neutralizing activity on day 28 after the first dose (NT50= 501, 1 week after the second dose), while there was a continual decrease until day 280. NT50 values further decreased to 51 by day 280 when approximately 85% of the participants had NT50 values of ,100 and approximately 36% had less than 20 NT50 or undetectable (Supplementary Figure 1). However, 2 weeks after administration of the third dose (205 participants [91.1%] remained in the cohort on day 300), there was a substantial rise in neutralizing activity, achieving an average NT50 of 3531. There was a concern that individuals who poorly responded to the second dose might again fail to produce sufficient neutralizing antibodies. Therefore, we specifically determined neutralization activity in vaccinees, who had achieved the lowest 10% level of neutralization following the second dose (n= 22, average-NT50= 110 on day 28; inset in Supplementary Figure 1). Notably, by day 300, all these low responders achieved markedly greater levels of neutralizing activity with an average NT50 of 2341 (range 482–9113 in VeroE6; Table 1). In HeLa TMPRSS2 cells, sera from these low responders substantially neutralized SARS-CoV-2, Alpha, Beta, Gamma, and Delta (geometric mean [gMean] NT50= 1777, 1350, 480, 1015, and 959, respectively), but had only marginal activity against Omicron/BA.1 with a gMean-NT50 of 52 (range≤ 20–197; Table 1). The same sera had similar neutralization profiles in VeroE6 cells. On the other hand, sera from participants who achieved the highest 10% level of neutralization on day 300 (n= 22, average NT50= 10 885 in VeroE6) had high neutralizing activity against SARS-CoV-2, Alpha, Beta, Gamma, and Delta (gMean NT50= 9774, 4906, 2279, 3271, and 3377, respectively) in HeLa TMPRSS2 cells and good neutralizing activity against Omicron/BA.1 (gMean NT50= 500, range 171–979). Notably, all day 280 sera of the highest 10% of participants failed to neutralize both Omicron/BA.1 and BA.2 (NT50 values ≤20; Supplementary Figure 2); however, these participants’ sera on day 300 also neutralized Omicron/BA.2 well (gMean-NT50= 702, range 262–1653; Table 1). The present data clearly show that a third dose of BNT162b2 elicits high-level SARSCoV-2-neutralizing antibodies even in those who poorly responded to the second dose, although low responders to the vaccines may be vulnerable to infection with Omicron sublineages BA.1 and BA.2. Of note, however, in terms of the effectiveness of a third dose of BNT162b2 against Omicron sublineages, the morbidity and mortality have yet to be determined between individuals who received the third dose but contracted symptomatic Omicron-related COVID-19 and those not receiving the third dose who contracted symptomatic Omicron-related COVID-19.

TO THE EDITOR—We read with interest the article by Mwimanzi et al about humoral responses after second and third doses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA vaccination in older adults [1]. Here, we determined the effects of consecutive 4 BNT162b2 doses on neutralizing activity in sera from participants aged ≥60 years and those with risk factors against SARS-CoV-2 Wuhan and 2 Omicron sublineages, and analyzed longitudinal changes of neutralization activity pre– and post– second through fourth BNT162b2 doses in a prospective study over 490 days. In the present prospective clinical study, 32 of 225 healthcare workers in Kumamoto General Hospital, Japan (225 were initially recruited in the primary clinical study [2]), who were either ≥60 years of age and/or had preexisting diseases/risk factors (see demographic characteristics in Supplementary Table 1A) were enrolled. These 32 participants received a fourth BNT162b2 dose as they had risk of developing severe coronavirus disease 2019. The SARS-CoV-2 neutralizing activity (50% neutralization titer [NT50]) of their sera against the Wuhan strain of SARS-CoV-2 was determined over 490 days using sera collected consecutively on (i) 1 week post–second dose, (ii) 2 weeks post–third dose, (iii) 1 week pre– fourth dose, and (iv) 2 weeks post–fourth dose (Supplementary Methods), representing a continuation of our previous studies [2–4]. We also evaluated the profile of S1-binding immunoglobulin G (IgG) levels following pre–/post–fourth dose (on days 300/490). In addition, we determined NT50 of the same sera using VeroE6 cells against infectious Omicron BA.2 and BA.5 variant sublineages, whose emergence has been associated with the present explosive increases globally [5]. Against SARS-CoV-2 Wuhan, moderate neutralizing activity was seen on day 28, 1 week after second-dose samples (Figure 1A, mean NT50 = 307), whereas there was a remarkable rise in neutralizing activity of the same participants’ sera of 2 weeks after third-dose administration (day 300), achieving a mean NT50 of 2238 (Figure 1A). On day 470 (1 week pre–fourth dose), the mean NT50 decreased to 541, 24% of the peak value on day 300 (2 weeks post–third dose) (Figure 1A). However, by day 490, 2 weeks post–fourth dose, neutralization activity was restored to 2096 (Figure 1A). The mean S1-binding IgG levels in pre–/post–fourth dose sera also showed an increase, from 247 on day 470 (1 week pre–fourth dose) to 1152 on day 490 (2 weeks post–fourth dose) (Supplementary Table 1B). On the other hand, mean NT50 values against BA.2 and BA.5 on day 28, 1 week post–second dose were only 27 and 24, respectively, close to the cutoff value (<20) (Figure 1B). By contrast, mean NT50 of sera on day 300 (2 weeks post–third dose) against BA.2 and BA.5 increased to 223 and 191, respectively (Figure 1B). By day 470, 1 week pre–fourth dose, the mean NT50 had decreased to 28% and 25% of those observed on day 300 sera against BA.2 and BA.5, respectively (Figure 1B, mean NT50 = 62 and 47, respectively); however, the mean NT50 against BA.2 and BA.5 was restored to 292 and 205, respectively, on day 490, to the extent seen after the third dose (Figure 1B). Compared to the significantly boosted response elicited by the third dose, the magnitudes of neutralizing activity against SARS-CoV-2 Wuhan following the fourth dose were only comparable to and not greater than those following the third dose (Figure 1). The magnitudes of neutralizing activity against 2 Omicron variants following the fourth dose were also comparable to and not greater than those following the third dose. The present data, that a fourth vaccine dose restores protection but does not further enhance the humoral response, may be related to “original antigenic sin” [6], wherein high-affinity memory B cells inhibit the recruitment of naive B cells against subsequent antigenic stimuli, in particular, against new stimuli. Thus, it is likely that despite the fourth dose, breakthrough infections continue to occur. It remains to be determined whether the upcoming Omicron-specific booster vaccines strengthen or attenuate the Omicronspecific protection already acquired through 4 regular doses in older individuals and those with other risks.

To date, there are no specific treatment regimens for HIV-1-related central nervous system (CNS) complications, such as HIV-1-associated neurocognitive disorders (HAND). Here, we report that two newly generated CNS-targeting HIV-1 protease (PR) inhibitors (PIs), GRL-08513 and GRL-08613, which have a P1-3,5- bis -fluorophenyl or P1- para -monofluorophenyl ring and P2-tetrahydropyrano-tetrahydrofuran ( Tp -THF) with a sulfonamide isostere, are potent against wild-type HIV-1 strains and multiple clinically isolated HIV-1 strains (50% effective concentration [EC 50 ]: 0.0001 to ∼0.0032 μM).