The COVID-19 pandemic has affected humans of all ages and conditions across the globe (Hu et al., 2021). Thanks to the rapid development of vaccines, a broad population immunity, at least against lethal variants, has been achieved in many countries (Watson et al., 2022). Nevertheless, even the most efficacious mRNA vaccines, despite achieving a significant reduction in morbidity and mortality, are failing to prevent new pandemic waves and it is uncertain what the level of protection will be against future viral genotypes (Andrews et al., 2022; Kirsebom et al., 2022).

Non-Pharmaceutical Interventions (NPI) including face masks, social distancing, individual isolation or lockdowns have shown to be effective in reducing the reproduction number (R) of SARS-CoV-2 (Li et al., 2021). Although lack of compliance with NPI in the first waves of the pandemic has been associated with significant production loss (Ohrnberger et al., 2021), the real-life impact of loss of active workforce in non-essential jobs is uncertain therefore, treatments to reduce viral colonization in upper airways have also been proposed as complementary methods. RNA sequencing studies showed a particularly high expression of viral entry factors, with the most affected target being the angiotensin converting enzyme 2 (ACE2) receptor located in the nasal epithelium (Vieira Braga et al., 2019; Deprez et al., 2020; Sungnak et al., 2020). While these data were initially useful to understand both the rapid spread and predominantly pneumonic phenotype of COVID-19, they also underlined the importance of the nose as the entry site and potential primary replication reservoir.

Intranasal photodynamic therapy (PDT) is an antimicrobial strategy based on the activation of a photoactive methylene blue-based compound within the anterior nares by a cold laser light (Zolfaghari et al., 2009). Nasal decolonization of Staphylococcus aureus via PDT has been shown to be a safe and effective strategy to minimize the risk of surgical site infections (Bryce et al., 2014). Recent reports suggest important anti-SARS-CoV-2 activity in ex vivo models (Lobo et al., 2022) and extensive in vitro PDT testing against wild strains of coronaviruses has been recently published (Arentz and von der Heide, 2022). Very recently, a proof-of-concept feasibility and safety study in humans within different hospital settings has been conducted showing signals of decay in infectivity assays (Pires et al., 2022).

We hypothesized that an early eradication of viral particles from the anterior nares by nasal-PDT could reduce viral spread and infectivity in SARS-CoV-2 patients and that photochemical internalization (PCI) of post-treatment residual antigens into dendritic cells might impact on patients’ immune responses.

Seventy-nine patients were screened between December 2021, and February 2022. One was excluded for not meeting the inclusion criteria. Of the 78 patients randomized 1:1, two patients abandoned the treatment group (one for local irritation, the other for personal reasons), and one patient abandoned the placebo group (for personal reasons). Seventy-five patients completed the study and were included in the analyses. Retention rate was 96.1% ( Figure 1 ).

The study-population consisted of mostly young healthy adults, with mild symptoms, naïve to natural SARS-CoV-2 infection (79%) with a complete course of vaccination (93%), and a high viral load ( Table 1 ).

Patients in the PDT arm showed a significant decrease in the in vitro infectivity measured by day 2 (Median of decrease: 61.75 (IQR: 0.14-1586) p<0.0001), while placebo patients did not (Median of decrease: 0.08 (IQR: -3.92-1150) p=0.24) ( Figure 2A ). Both arms demonstrated a significant decrease in infectivity by day 7. ( Figure 2A ). In the multi-adjusted linear regression model, PDT-treatment demonstrated a protective effect at 3 days with a mean β coefficient of -812 (95%CI -478660- -1.3, p<0.05) and a trend at 7 days with a mean β coefficient of -9.3 (95%CI -269.1- 3.1, p NS) ( Table 2 ).

During the study, both groups presented increased PCR cycle counts, although PDT-treated patients showed a significant difference in both E and N genes at day 7. ( Figure 2B ). When analysing the probability of being PCR positive at day 7, nasal PDT showed a protection against PCR positivity with Odds Ratio of 0.16 (95% CI: 0.05-0.52) and 0.15 (95% CI: 0.04-0.58), using PCR thresholds of 32 or 34 cycles respectively ( Table 2 ; Figure 2C ).

Antibody quantification at 10 and 20 weeks showed no differences in the production of anti-spike ( Figure 3A ) and anti-nucleocapsid ( Figure 3B ) antibodies, between control and PDT arms ( Supplementary Table 1 ). There was a significant decay in Anti-Spike antibodies at 20 weeks in the placebo group (p=0.001), but not in the PDT group (p= 0. 0859) ( Figures 3A, C, D ). The measurement of interferon (IFN) synthesis by CD4 T cells after exposure to SARS-CoV-2 spike antigen revealed lower median IFN units in the placebo group when compared to PDT. ( Supplementary Table 2 ; Figure 4A ). CD4 and CD8 T-cell responses against the Spike protein were also trending higher in PDT-treated individuals with medians at 20 weeks almost double than placebo (p = 0.0971) ( Supplementary Table 2 ; Figure 4B ). When CD4 and CD8 cells were exposed to SARS-CoV-2, a trend for increased T-cell responses were observed in the PDT group when compared to placebo, at 10 and 20 weeks (p = 0.0916 and p = 0.0550) ( Supplementary Table 2 ; Figure 4C ). The decay of specific T-cell immunity against SARS-CoV-2 from week 10 to 20 tended to be of higher significance in the control group (p = 0.0593), than in the PDT group (p = 0.232) ( Figures 4C–E ).

COVID-19 RNA sequencing was successful in 72 patients (96%). The results indicate that 97.2% of patients were infected with Omicron 21K variant (B.1.1.529 or BA.1) one with Omicron 21L (BA.2) and one with Delta 21J ( Supplementary Figure 1 )

When analysing the progression of symptoms at day 3 and 7, a significant decrease in chest tightness and headache was observed in the PDT-treated group ( Table 3 ).

Regarding intervention safety, 32 patients (5 in the control group and 27 in the intervention group) reported a total of 53 mild adverse events. One patient in the intervention group dropped out due to local itching after first PDT ( Supplementary Figure 2 ).

A 3-day treatment with intranasal PDT can reduce SARS-CoV-2 infectivity and induce specific T-cell responses in COVID-19 patients. When applied in early stages of SARS-CoV-2 infection, local decolonization of nasal passages could be clinically relevant for the inhibition of viral spread both from person to person and from upper to lower respiratory tract, in clinical and non-clinical settings. PDT has been proposed as a valid approach to inactivate SARS-CoV-2 (Almeida et al., 2020; Dias et al., 2020; Sabino et al., 2020; Ziganshyna et al., 2022), and the ex-vivo efficacy of PDT against SARS-CoV-2 virion has been demonstrated (Arentz and von der Heide, 2022). This study is the first prospective randomized trial exploring the safety and efficacy of PDT nasal decolonization in SARS-CoV-2 infected individuals.

The primary endpoint of the trial (infectivity reduction) was achieved. We demonstrated a reduction of infectivity in mildly symptomatic, otherwise-healthy individuals with high viral load (Ct<27), when applied during the first days of the replication phase in the nasal mucosa. The treatment consisted of three consecutive day, 12 min-applications of methylene blue-based PDT. Because of the absence of prior literature in intranasal PDT treatment of SARS-CoV-2, we decided to adopt a repeated application protocol rather than the single- or double-applications tested in vitro. This was supported by the low toxicity of the technique, the expected high viral replication rate, the intracellular life of viruses and the interference of mucous layers, especially in patients with nasal congestion. Whether an intensified protocol could demonstrate further-enhanced efficacy while maintaining acceptable local toxicity, should be explored in future studies. Interestingly, although the treatment with PDT was applied only to the nasal passages, it also reduced SARS-CoV-2 colonization of pharyngeal region. This result strengthens the hypothesis of the importance of early nasal decolonization in a disease that could develop to a systemic stage in few days.

The effects of PDT were observed as soon as 3 days after treatment using an in vitro infectivity assay with NP swabs. PCR thresholds were also higher in the treated group at day 7, a further sign of the durable effect of nasal decolonization. Consequently, PDT treatment demonstrated an important protective effect against PCR positivity. There were no serious adverse effects reported during treatment.

We observed a sharp decrease in viral load in our entire population (placebo and PDT) between day 3 and 7, which presumably masked the true magnitude of the PDT. This was probably related to the fact that vaccination is associated with faster viral RNA decline (Kissler et al., 2021; Chia et al., 2022) and that almost all patients were infected by Omicron variant (21K, BA.1) (97.2%) with a favourable biology (Bouzid et al., 2022). Despite these two masking effects, patients in the PDT arm demonstrated significantly higher elimination rates of viral particles.

The titer of anti-S and anti-N antibodies at 10 and 20 weeks after treatment was not different between the control and the intervention group. In both groups the amount of anti-S and anti-N antibody decreased at 20 weeks, revealing the decline of humoral response already described in other studies of SARS-CoV-2 infection (Fernandez-Ciriza et al., 2022). However, at week 20, the decrease in anti-S antibody was significant only in the control group. This could suggest a deceleration of the natural decline of specific B-cell responses by PDT treatment, although this concept should be confirmed in future studies with larger cohorts. Although the median decrease of anti-N antibody was also higher in the placebo group than in the PDT group, this did not reach statistical significance due to the large dispersion of the data in both groups.

Similar to what we found in the humoral response, no significant differences were seen in the specific T-cell immunity against SARS-CoV-2 between control and intervention groups. Paralleling the humoral response, subjects in both groups experienced a reduction of the specific T cell responses between the week 10 and 20. Interestingly, the decline was significant only in placebo-treated patients for both CD4 and CD4+CD8 anti-S T-Cell responses, but not in PDT-treated subjects ( Figures 4A, B ; Supplementary Table 2 ). Regarding the CD4+CD8 anti-whole SARS-CoV-2 genome the median interferon produced by T Cells was superior in PDT-treated subjects, with trending p values bot at 10 weeks and at 20 weeks ( Figure 4C and Supplementary Table 2 ). The relevance of the effect of nasal PDT treatment on preventing the natural decline and/or inducing T cell immunity against SARS-CoV-2, has not been described so far. One hypothesis is that the destruction of viral particles in the nasal mucosa, could enhance the uptake, processing, or presentation of viral peptides by Antigen Presenting Cells (APC). In the field of cancer vaccines, the use of PDT enhanced adjuvant-free cross-priming of APC to CD8 T lymphocytes (Mroz et al., 2010). In a β-galactosidase-bearing colon adenocarcinoma model, the use of vascular PDT induced the remission of tumors. T lymphocytes isolated from cured mice recognized the β-galactosidase-derived epitopes and kill only cancer cells expressing β-galactosidase. The anti-tumoral response to PDT treatment was able to eliminate distant untreated antigen-expressing lesions (Huang et al., 2012). These data suggest that PDT could also induce APC uptake of SARS-CoV-2 antigens in the nasal mucosa of infected patients. Future preclinical studies should be directed to test this hypothesis. If confirmed, this effect of PDT may be of great relevance in reducing transmission of future viral variants, where immune escape is otherwise almost certain.

Other studies with different local treatments have been performed. A double-blind, randomized, placebo-controlled trial in patients with mildly symptomatic SARS-CoV-2 infections (n=306) of treatment with nitric oxide nasal spray (NONS) was evaluated. NONS was self-administered six times daily for 7 days (Schikora et al., 2020). This work had several similarities with our study: both evaluated inactivation of nasal viral load through local treatments to shorten infectivity, and both utilized a similar population of non-severe SARS-CoV-2 patients. However, the patients in the NONS trial were only tested by RT-PCR while our study used both RT-PCR and infectivity assay, increasing both sensitivity and relevance to downstream transmission (Jamal et al., 2021; Berenger et al., 2022). Second, the NONS study included ~ 60% unvaccinated patients and did not include follow-up, while our study included follow-up visits to demonstrate absence of viral replication rebound. Finally, the impact of the NONS treatment on anti-SARS-CoV-2 immunity is unknown, whereas this was convincingly demonstrated in our PDT study.

The present study had several limitations, the power of the study was planned for the infectivity assay, but full recruitment was unable to be completed due to the decrease in SARS-CoV-2 cases in February 2022 in Spain. The study was not powered to detect differences in the IFN assays, but based on the differences observed between groups, a higher sample size would probably reveal consistently significant results. Finally, the present study only incorporated individuals infected with the Omicron strain. In future studies, to better characterize the immunological effect of PDT in clinical setting, flow cytometry studies could be used to describe the activation of innate immune cells (including NK and Dendritic Cells) and the specific phenotype of CD4 T lymphocytes (naïve, regulatory or memory T cells), before and after PDT treatment. In addition, circulating cytokines and chemokines, especially at short times, could help in defining the immune landscape of these patients, and whether the treatment with PDT can alter it.

Strengths of the study included the high retention rate, the follow-up period and the infectivity assays along with humoral and cellular immunity evaluations up to 20 weeks after infection, all of which help to better elucidate the effect of nasal PDT on SARS-CoV-2 colonization and immunization. The impact of PDT in different settings such as ENT or dentistry clinics, Intensive Care Units, first responder settings, or GI laboratories where contact with potentially infectious patients could jeopardize efficiency of diagnostic and therapeutic procedures warrants further investigation. Development of new affordable and wearable nasal illuminators might provide an alternative, low-cost, widely accessible, respiratory virus treatment during future pandemics.

PDT treatment was well tolerated, and no serious adverse effects were detected. Nasal PDT enhances SARS-CoV-2 viral clearance in mild COVID-19 patients, even in vaccinated individuals, rapidly decreasing infectivity. Nasal PDT decelerates the decline of SARS-CoV-2 specific T-cell immune responses in vaccinated individuals infected with Omicron variant.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Institutional Review Board of the University of Navarra (registry number: EC_2021/1). The patients/participants provided their written informed consent to participate in this study.

JA, FC-T and AF-M conceived the study, designed the protocol, submitted the requested changes the Institutional Board Review for its approval, organized the field work, discussed the results, and wrote the manuscript. JK helped on the trial design. AF-M recruited and randomized the patients. JZ, MP-S, LM-N, NM-R, MP, MM-F treated and followed patients. MR organized and performed the RT-PCR studies. GR organized and performed the immunological assays. JP and FC-T reviewed the microbiological and immunological data. JQ, CS, GG-A, LM-G, and JP reviewed the manuscript. CS, SM, CO, GC, JR. and GG-A coordinated and performed the in vitro infectivity assays. FM, JZaz and MPoz prepared the libraries, did the sequencing and performed variant analysis. All authors contributed to the article and approved the submitted version.