Omicron BA.4/BA.5 escape neutralizing immunity elicited by BA.1 infection

We isolated live BA.4 and BA.5 viruses from infections in South Africa to test against pre-existing immunity. This consisted of sera from unvaccinated (n = 24) and vaccinated (n = 15) people infected in the preceding infection wave which was BA.1 dominated (Fig. 1a). This cohort was previously described by us21 and consisted of participants with mostly mild Omicron BA.1 infections who were sampled weekly from symptom onset. Samples used here were collected a median of 23 days (IQR 19–27 days) post-symptom onset, once the participants developed or increased their BA.1 neutralizing response21. We also tested the viruses against sera from people who were vaccinated but not BA.1 infected (n = 18, see Supplementary Table 1 for cohort details). For study participants infected in the Omicron BA.1 infection wave, the majority (25 out of 39 infections) were confirmed Omicron/BA.1 by sequencing the infecting virus21 (Table S1).

To quantify neutralization, we report the 50% focus reduction neutralization test value (FRNT50), which is the inverse of the plasma dilution required for a 50% reduction in the number of infection foci relative to the no antibody control in a live virus neutralization assay22.

We first report neutralization in the 24 unvaccinated study participants infected with BA.1. Neutralization of BA.1 was low at FRNT50 = 275. The FRNT50 declined to 36 for BA.4 and 37 for BA.5 neutralization (Fig. 1d), 7.5 and 7.6-fold drops, respectively relative to BA.1 neutralization (Fig. 1e).

In vaccinated participants with BA.1 breakthrough infection after vaccination, BA.4 and BA.5 neutralization dropped from 507 for BA.1 to 158 for BA.4 and 198 BA.5 (Fig. 2a). The corresponding fold-drops were 3.2 for BA.4 and 2.6 for BA.5 (Fig. 2b). Given that the vaccines were designed with ancestral SARS-CoV-2 sequence, neutralization capacity against the ancestral virus with the D614G substitution may be a second benchmark to measure escape in this group. We therefore compared the neutralization of the Omicron sub-lineages to neutralization capacity against an isolate of ancestral virus from the B.1 lineage containing the D614G substitution. Neutralization of this ancestral isolate had an FRNT50 of 2038, substantially higher than BA.1 neutralization by the same plasma (Fig. 2a). Compared to ancestral virus, neutralization dropped 4.0-fold for BA.1, 12.9-fold for BA.4, and 10.3-fold for BA.5 (Fig. 2c).

Fig. 2: Escape of BA.4 and BA.5 from immunity elicited by vaccination combined with BA.1 breakthrough infection.
figure 2

a Neutralization of ancestral virus with the D614G substitution, BA.1, BA.4 and BA.5 by vaccine elicited neutralizing immunity with BA.1 breakthrough infection in n = 15 participants. Numbers are geometric mean titer (GMT) FRNT50. Dashed line is most concentrated plasma tested. b Geometric mean (GM) of fold- drops in neutralization and their 95% confidence intervals for BA.4 and BA.5 relative to BA.1 calculated from (a). c GM of fold-drops in neutralization and their 95% confidence intervals for BA.1, BA.4 and BA.5 relative to ancestral/D614G virus calculated from (a). For all panels, green points are values for ancestral/D614G, orange points are BA.1, yellow points are BA.4, and pink points are BA.5. p-values were determined by a two-sided Wilcoxon rank sum test and represented as **0.01-0.001, ***0.001-0.0001, ****<0.0001. Exact p-values were 7.9 × 10−3 for BA.1, 9.7 × 10−5 for BA.4, and 1.9 × 10−4 for BA.5. Source data are provided as a Source Data file.

Because the cohort contained participants vaccinated with the Johnson and Johnson Ad26.CoV2.S in addition to the Pfizer BNT162b2 vaccine (Table S1) and participants who differed in their HIV-1 status (14 were people living with HIV, of whom 13 were virologically suppressed with antiretroviral therapy21), we examined whether HIV status and vaccine type impacted our results by comparing the fold-drop in neutralization of BA.4 and BA.5 to BA.1 in the different subgroups. Within the vaccinated group, the fold-drop with BA.4 and BA.5 was very similar when comparing neutralization of sera from participants vaccinated with Ad26.CoV2.S versus BNT162b2 (Fig. S1a). Likewise, fold-drops in neutralization did not substantially change between vaccinated people living with HIV and HIV negative participants (Fig. S1b). In contrast, there was a trend with borderline significance that showed higher BA.4 and BA.5 escape in people living with HIV who were unvaccinated (Fig. S1c).

The L452R and F486V mutations in the spike receptor binding domain could potentially mediate escape from vaccine elicited neutralization independently of BA.1 infection elicited immunity. To test this, we measured BA.1, BA.4, and BA.5 neutralization relative to ancestral D614G virus in 18 vaccinated South African participants who did not have BA.1 breakthrough infection (Table S1). Because we have previously observed that Beta variant infection may broaden vaccine elicited neutralization capacity23, we did not include participants previously infected with a variant and restricted this group to either individuals who were vaccinated only or vaccinated and infected with ancestral/D614G. Here neutralization declined from FRNT50 = 4123 for ancestral/D614G to 208 for BA.1, 211 for BA.4 and 197 for BA.5 (Fig. 3a). BA.4 and BA.5 neutralization did not drop compared to BA.1 in this group (Fig. 3b). Fold-drops relative to ancestral virus were 19.8-fold for BA.1, 19.6-fold for BA.4 and 20.9-fold for BA.5 (Fig. 3c).

Fig. 3: Escape of BA.4 and BA.5 from immunity elicited by vaccination combined in the absence of BA.1 infection.
figure 3

a Neutralization of ancestral/D614G, BA.1, BA.4 and BA.5 by vaccine elicited neutralizing immunity in n = 18 participants. Numbers are geometric mean titer (GMT) FRNT50. Dashed line is most concentrated plasma tested. b Geometric mean (GM) of fold-drops in neutralization and their 95% confidence intervals for BA.4 and BA.5 relative to BA.1 calculated from (a). c GM of fold-drops in neutralization and their 95% confidence intervals for BA.1, BA.4 and BA.5 relative to ancestral/D614G virus calculated from (a). For all panels, green points are values for ancestral/D614G, orange points are BA.1, yellow points are BA.4, and pink points are BA.5. p-values were determined by a two-sided Wilcoxon rank sum test and represented as ****<0.0001. Exact p-values were 7.2 × 10−5 for BA.1, 3.2 × 10−5 for BA.4, and 2.4 × 10−5 for BA.5. Source data are provided as a Source Data file.

We observed that escape of BA.4 and BA.5 relative to BA.1 from neutralizing immunity was strongest in BA.1 infected unvaccinated individuals (Fig. 1e) and was moderated by vaccination in vaccinated people with BA.1 breakthrough infection (Fig. 2b). In contrast, BA.1, BA.4, and BA.5 showed similar (and extensive) escape in vaccinated people who did not have BA.1 infection elicited immunity (Fig. 3b). 

BA.4 and BA.5 viruses showed very similar neutralization escape to each other, with minor differences which may be explained by experimental variation. This is expected since they share the same spike sequence, with the exception that our BA.4 isolate contained the N658S spike mutation found in a subset of BA.4 sequenced infections reported in GISAID (27% at the time of writing, see https://outbreak.info/compare-lineages?pango=BA.4&gene=S&threshold=10&nthresh=1&sub=false&dark=false) but not in BA.5. However, because we test neutralization against the live virus and not spike alone, we cannot rule out that the difference is real and occurs because of differences in the other genes (which may perhaps modulate neutralization by influencing spike surface expression or another parameter not directly related to spike sequence). In contrast to BA.4 and BA.5, we detected only minor escape of BA.2 from BA.1 elicited immunity in the same cohort of BA.1 infected individuals in a previous study21.

As we previously reported21 and confirmed here, BA.1 elicits relatively weak neutralization in the absence of vaccination, consistent with reports showing that Omicron has reduced immunogenicity24,25,26. Even with BA.1 breakthrough infection, the FRNT50 against ancestral virus was about half of that measured in a group composed mostly (Table S1) of people with ancestral infection and vaccination hybrid immunity (Fig. 3a). However, there are caveats to this comparison, including the order of infection and vaccination, with infection occurring first in the non-BA.1 infected group and the samples collected after vaccination.

Since our original release of the BA.4 and BA.5 neutralization results, other groups reported similar conclusions27,28,29,30,31, with BA.4 and BA.5 escape from BA.1 and BA.2 elicited immunity being very similar to our measurements. These studies analyzed different cohorts from us and from each other, yet the results converged. Our cohort, which enrolls people who use the South African public health system, is generally distinguished from cohorts in other countries with active sero-surveillance of variants by the higher proportion of people who are unvaccinated, the higher proportion of people vaccinated with the Johnson and Johnson Ad26.CoV2.S vaccine, and the higher proportion of people who are living with HIV. In the vaccinated group we did not find evidence that either vaccine type or HIV status impacted the fold-drop in neutralization observed with BA.4 and BA.5 relative to BA.1. However, there was a trend to higher escape of BA.4 and BA.5 in unvaccinated individuals living with HIV. This is consistent with our previous results showing that the neutralization response elicited by a vaccine to a variant is similar between people living with HIV who are well suppressed with antiretroviral therapy and people who are HIV-negative, but that the response is attenuated by HIV in unvaccinated people32.

A recent report showed that BA.4/BA.5 was more fusogenic in cell culture and more pathogenic relative to BA.2 in the hamster model27. However, despite this the BA.4/BA.5 infection wave in South Africa did not lead to a sharp increase in excess deaths associated with the other infection waves, although the association was also reduced in the BA.1 infection wave (Fig. 1a). This may indicate that, while SARS-CoV-2 pathogenicity continues to fluctuate and may evolve away from the attenuated pathogenicity observed in BA.133, the increased population immunity may keep disease severity relatively low34. Consistent with this, a recent analysis showed that neutralization capacity required to prevent severe disease is considerably lower than that required to prevent symptomatic infection35. In addition, there may be factors specific to the South African infection environment which reduce pathogenicity such as immunity from Beta infection combined with vaccination, which we found to broaden neutralization capacity against BA.4 and BA.523.

Limitations of this study include that we did not have enough participants with BA.2 infection or booster vaccination to test escape against this type of elicited immunity, which is much more common in some countries, for example those in Europe and North America. Our cohort is heterogeneous in terms of vaccination. Most participants are not vaccinated. Vaccinated participants are divided into two almost equal groups of Pfizer BNT162b2 and Johnson and Johnson Ad26.CoV2.S, though when we compared these vaccinated groups we observed that they were similar in terms of BA.4 and BA.5 escape. This may raise concerns that the heterogeneity in the relatively small vaccinated group may limit our ability to make more general conclusions about the degree of BA.4 and BA.5 immune escape in BA.1 infected vaccinated individuals.

Furthermore, the South African population differs from that of other countries where SARS-CoV-2 infection is intensively studied. South Africa has a lower fraction of vaccinated people, higher HIV prevalence, and people with previous immunity from an extensive Beta variant infection wave22,23,36. Every cohort is specific to the population it is drawn from, and it takes cohorts from multiple countries to get an accurate measure of immune escape of variants globally. The heterogeneity of individuals in our cohort reflects the heterogeneity in the South African population, and we chose not to limit our investigation to a specific subgroup. What may be specifically relevant in the population we study is that BA.4/BA.5, as well as BA.1, were first detected in South Africa and likely evolved in this region. Therefore, our study may indicate the selective forces at play in BA.4/BA.5 evolution. Given our observation that BA.4 and BA.5 have the strongest neutralization escape advantage in unvaccinated people, it may be important to determine whether the increasing vaccination coverage will reduce variant evolution.

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